U.S. patent application number 11/770447 was filed with the patent office on 2007-11-15 for genes of an otitis media isolate of nontypeable haemophilus influenzae.
This patent application is currently assigned to CHILDREN'S HOSPITAL INC.. Invention is credited to Lauren O. Bakaletz, David W. Dyer, Robert S. JR. Munson.
Application Number | 20070264256 11/770447 |
Document ID | / |
Family ID | 32962758 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264256 |
Kind Code |
A1 |
Bakaletz; Lauren O. ; et
al. |
November 15, 2007 |
Genes of an Otitis Media Isolate of Nontypeable Haemophilus
Influenzae
Abstract
The invention relates to the polynucleotide sequence of a
nontypeable stain of Haemophilus influenzae (NTHi) and polypeptides
encoded by the polynucleotides and uses thereof. The invention also
relates to NTHi genes which are upregulated during or in response
to NTHi infection of the middle ear and/or the nasopharynx.
Inventors: |
Bakaletz; Lauren O.;
(Hilliard, OH) ; Munson; Robert S. JR.; (Hilliard,
OH) ; Dyer; David W.; (Oklahoma City, OK) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
CHILDREN'S HOSPITAL INC.
Columbus
OH
The Board of Regents of University of Oklahoma
Norman
OK
|
Family ID: |
32962758 |
Appl. No.: |
11/770447 |
Filed: |
June 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10795159 |
Mar 5, 2004 |
7241867 |
|
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11770447 |
Jun 28, 2007 |
|
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60453134 |
Mar 6, 2003 |
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Current U.S.
Class: |
424/130.1 ;
424/190.1; 435/7.32; 514/2.8; 514/44A; 530/350; 530/387.1;
536/23.1 |
Current CPC
Class: |
A61P 27/16 20180101;
C12Q 1/689 20130101; A61P 31/12 20180101; A61P 37/04 20180101; G01N
33/56911 20130101; A61P 31/04 20180101; A61K 38/00 20130101; C07K
16/1242 20130101; C07K 14/285 20130101; A61P 31/00 20180101; A61K
39/00 20130101; C12N 15/113 20130101 |
Class at
Publication: |
424/130.1 ;
424/190.1; 435/007.32; 514/002; 514/044; 530/350; 530/387.1;
536/023.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/00 20060101 A61K038/00; A61K 48/00 20060101
A61K048/00; C07K 14/00 20060101 C07K014/00; G01N 33/53 20060101
G01N033/53; C07K 16/18 20060101 C07K016/18; C07H 21/00 20060101
C07H021/00; A61K 39/00 20060101 A61K039/00 |
Claims
1. An isolated polynucleotide comprising the nucleotide sequence of
any one of SEQ ID NOS:577-579, SEQ ID NOS: 589-614, SEQ ID NOS:
675-685, SEQ ID NO: 615, SEQ ID NO: 617, SEQ ID NO: 619, SEQ ID NO:
621, SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 627, SEQ ID NO:
629, SEQ ID NO: 631, SEQ ID NO: 633, SEQ ID NO: 635, SEQ ID NO:
637, SEQ ID NO: 639, SEQ ID NO: 641, SEQ ID NO: 643, SEQ ID NO:
645, SEQ ID NO: 647, SEQ ID NO: 649, SEQ ID NO: 651, SEQ ID NO:
653, SEQ ID NO: 655, SEQ ID NO: 657, SEQ ID NO: 659, SEQ ID NO:
661, SEQ ID NO: 663, SEQ ID NO: 665, SEQ ID NO: 667, SEQ ID NO:
669, 671, SEQ ID NO: 673, SEQ ID NO: 686, SEQ ID NO: 688, SEQ ID
NO: 692, SEQ ID NO: 694, SEQ ID NO: 696, SEQ ID NO: 698, SEQ ID NO:
700, SEQ ID NO: 702, SEQ ID NO: 704, SEQ ID NO: 706, SEQ ID NO:
708, SEQ ID NO: 710, SEQ ID NO: 712, SEQ ID NO: 714, SEQ ID NO:
716, SEQ ID NO: 718, SEQ ID NO: 720, SEQ ID NO: 722, SEQ ID NO:
724, SEQ ID NO:726, SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732,
SEQ ID NO: 734, SEQ ID NO: 736, SEQ ID NO: 738, SEQ ID NO: 740, SEQ
ID NO: 742, SEQ ID NO: 744, SEQ ID NO: 746, SEQ ID NO: 748, SEQ ID
NO: 750, SEQ ID NO: 752, SEQ ID NO: 754, SEQ ID NO: 756, SEQ ID
NO:758, SEQ ID NO:760, SEQ ID NO: 762, SEQ ID NO: 764, SEQ ID NO:
766, SEQ ID NO:768 or SEQ ID NO: 770.
2. An isolated polypeptide comprising an amino acid sequence
encoded by a nucleotide sequence set of claim 1 or a fragment
thereof.
3. An isolated polypeptide comprising an amino acid sequence of any
one of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO:,620, SEQ ID NO:
622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO:
628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO:
636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO:
644, SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO:
652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO:
660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO:
668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO:
687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO:
695, SEQ ID NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO:
703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO:
711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719,
SEQ ID NO:721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID
NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO:
737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO:
745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO:
753, SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO:
761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID
NO: 771.
4. A composition comprising a polypeptide of claim 2 or 3 and a
pharmaceutically acceptable carrier.
5. An antibody that specifically binds to a polypeptide of claim 2
or 3 or fragment thereof.
6. A composition comprising an antibody of claim 5 and a
pharmaceutically acceptable carrier.
7. A method for detecting NTHi bacteria in a biological sample
comprising (a) contacting a polynucleotide of claim 1 or a fragment
thereof with a biological sample, and (b) detecting hybridization
of the polynucleotide within the sample.
8. A method for detecting NTHi bacteria in a biological sample
comprising: (a) contacting an antibody of claim 5 with a biological
sample, and (b) detecting binding of the antibody within the
sample.
9. The method of claim 7 or 8 wherein the biological sample is
selected from the group consisting of serum, sputum, ear fluid,
blood, urine, lymphatic fluid, and cerebrospinal fluid.
10. A method for eliciting an immune response to NTHi bacteria
comprising administering an immunogenically effective dose of a
polypeptide of claim 2 or 3 or a fragment thereof to a patient at
risk of NTHi bacterial infection.
11. A vaccine comprising a polypeptide of claim 2 or 3 or a
fragment thereof and a pharmaceutically suitable carrier.
12. A method for preventing NTHi bacterial infection comprising
administering a polypeptide of claim 2 or 3 that blocks cellular
attachment of NTHi bacteria to a patient at risk of NTHi bacterial
infection.
13. A method for preventing NTHi bacterial infection comprising
administering an antibody of claim 5 that blocks cellular
attachment of NTHi bacteria to a patient at risk of NTHi bacterial
infection.
14. The method of claim 13 wherein the NTHi infection is in the
middle ear.
15. A method of treating or preventing NTHi bacterial infection
comprising administering a molecule that inhibits expression or
activity of a polypeptide of claim 2 or 3 to an patient in
need.
16. The method of claim 15 wherein the NTHi polypeptide comprises
the is encoded by an NTHi gene selected from the group consisting
of hisB, lppB, sapA, rbsC, pure, rib, arcB, uxuA, licC, ispZ, mukF,
glpR, ihfB, cspD, lav, HI1647, HI0094, HI1163, HI0665, HI1292,
HI1064, HI1386, HI1462, HI1369, and HI1598.
17. The method of claim 15 wherein the NTHi polypeptide comprises
the amino acid sequence selected from the group consisting of SEQ
ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID
NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 634, SEQ ID NO:
638, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO:
650, SEQ ID NO: 652, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO:
660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO:
668, SEQ ID NO: 670, SEQ ID NO:672 and SEQ ID NO: 674.
18. The method of claim 15 wherein the molecule administered to the
patient in need is an antisense oligonucleotide.
19. The method of claim 15 wherein the molecule administered to the
patient in need is an antibody.
20. The method of claim 15 wherein the molecule administered to the
patient in need is a small molecule.
21. The method of claims 15 wherein the NTHi infection is in the
middle ear.
Description
RELATED APPLICATIONS
[0001] The present application claims priority benefit from U.S.
Provisional Application 60/453,134 filed Mar. 6, 2003 which is
incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0002] The invention relates to the polynucleotide sequence of a
nontypeable strain of Haemophilus influenzae (NTHi) genome, NTHi
genes contained within the genome and polypeptides encoded by the
polynucleotides. The invention also relates to uses of these NTHi
polynucleotides and NTHi polypeptides including vaccines and
methods of treating and preventing NTHi related disorders. The
invention also relates to NTHi genes which are upregulated during
or in response to NTHi infection of the middle ear or
nasopharynx.
BACKGROUND
[0003] Otitis media (OM) is a highly prevalent pediatric disease
worldwide and is the primary cause for emergency room visits by
children (Infante-Rivand and Fernandez, Epidemiol. Rev., 15:
444-465, 1993). Recent statistic indicate that 24.5 million
physician office visits were made for OM in 1990, representing a
greater than 200% increase over those reported in the 1980's. While
rarely associated with mortality any longer, the morbidity
associated with OM is significant. Hearing loss is a common problem
associated with this disease, often times affecting a child's
behavior, education and development of language skills (Baldwin,
Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol.
Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis.,
162: 685-694, 1990). The socioeconomic impact of OM is also great,
with direct and indirect costs of diagnosing and managing OM
exceeding $5 billion annually in the U.S. alone (Kaplan et al.,
Pediatr. Infect. Dis. J., 16: S9-11, 1997).
[0004] Whereas antibiotic therapy is common and the surgical
placement of tympanostomy tubes has been successful in terms of
draining effusions, clearing infection and relieving pain
associated with the accumulation of fluids in the middle ear, the
emergence of multiple antibiotic-resistant bacteria and the
invasive nature associated with tube placement, has illuminated the
need for more effective and accepted approaches to the management
and preferably, the prevention of OM. Surgical management of
chronic OM involves the insertion of tympanostomy tubes through the
tympanic membrane while a child is under general anesthesia. While
this procedure is commonplace (prevalence rates are .about.13%;
Bright et al., Am. J. Public Health, 83(7): 1026-8, 1993) and is
highly effective in terms of relieving painful symptoms by draining
the middle ear of accumulated fluids, it too has met with criticism
due to the invasive nature of the procedure and its incumbent risks
(Berman et al., Pediatrics, 93(3):353-63, 1994; Bright et al.,
supra.; Cimons, ASM News, 60: 527-528; Paap, Ann. Pharmacother.,
30(11): 1291-7, 1996).
[0005] Progress in vaccine development is most advanced for
Streptococcus pneumoniae, the primary causative agent of acute OM
(AOM), as evidenced by the recent approval and release of a
seven-valent capsular-conjugate vaccine, PREVNAR.RTM. (Eskola and
Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). While
PREVNAR.RTM. has been highly efficacious for invasive pneumococcal
disease, coverage for OM has been disappointing (6-8%) with reports
of an increased number of OM cases due to serotypes not included in
the vaccine (Black et al., Pedriatr. Infect. Dis J., 19: 187-195;
Eskola et al., Pedriatr. Infect. Dis. J., 19: S72-78, 2000; Eskola
et al., N. Engl. J. Med. 344: 403-409, 2001; Snow et al., Otol.
Neurotol., 23: 1-2, 2002). Less progress has been made for
non-typeable Haemophilus influenzae (NTHi), the gram-negative
pathogen that predominates in chronic OM with effusion (Klein,
Pedriatr. Infect. Dis. J., 16: S5-8, 1997; Spinola et al., J.
Infect. Dis., 154: 100-109, 1986). Hampering development of
effective vaccines against NTHi, is the currently incomplete
understanding of the pathogenesis of NTHi-induced middle ear
disease. Contributing to this delay is a lack of understanding of
the dynamic interplay between microbe-expressed virulence factors
and the host's immune response as the disease progresses from one
of host immunological tolerance of a benign nasopharyngeal
commensal, to that of an active defensive reaction to an
opportunistic invader of the normally sterile middle ear space.
[0006] Currently there is a poor understanding of how NTHi causes
OM in children. The identification of putative virulence factors
necessary for induction of OM will contribute significantly to the
understanding of the host-pathogen interaction and ultimately, the
identification of potential vaccine candidates and targets of
chemotherapy. There is a tremendous need to develop more effective
and accepted approaches to the management and preferably, the
prevention of otitis media. Vaccine development is a very promising
and cost effective method to accomplish this goal (Giebank,
Pedriatr. Infect. Dis. J., 13(11): 1064-8, 1994: Karma et al., Int.
J. Pediatr. Otorhinolaryngol., 32(Suppl.): S127-34, 1995).
SUMMARY OF INVENTION
[0007] The present invention provides for the identification and
characterization of the genomic sequence of NTHi H. influenzae
strain 86-028NP and the polypeptide sequences encoded thereby. The
3-fold analysis of the NTHi genomic sequence is set out in a series
of contig sequences denoted as SEQ ID NO: 1-576, and the subsequent
8-fold analysis of the genomic sequence is set out in a series of
11 contig sequences denoted as SEQ ID NOS: 675-685. These contigs
are raw data and one of skill in the art may assemble these contigs
by comparing overlapping sequences to construct the complete genome
of the NTHi stain 86-028NP using routine methods.
[0008] The present invention also provides for antibodies specific
for the NTHi polypeptides of the invention. Methods of detecting
NTHi bacteria in a human or in sample, such as serum, sputum, ear
fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are
contemplated. These methods include detecting NTHi polynucleotides
with specific polynucleotide probes or detecting NTHi polypeptides
with specific antibodies. The invention also contemplates
diagnostic kits which utilize these methods of detecting NTHi
bacteria.
[0009] The present invention also contemplates methods of eliciting
an immune response by administering a NTHi polypeptide of the
invention or a NTHi peptide thereof. These methods include
administering the NTHi polypeptide or NTHi peptide as a vaccine for
treatment and/or prevention of diseases caused by NTHi infection,
such as OM. The following NTHi genes are upregulated during or in
response to middle ear and/or nasopharynx infections; and the
polypeptides encoded by these genes and peptides thereof are
contemplates as possible OM vaccine candidates and/or target of
chemotherapy: hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA,
dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD,
HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. NTHi hisB gene is
set out as nucleotide sequence SEQ ID NO: 615 and encodes the amino
acid sequence set out as SEQ ID NO: 616. NTHi sapA gene is set out
as nucleotide sequence SEQ ID NO: 617 and encodes the amino acid
sequence set out as SEQ ID NO: 618. NTHi rbsC gene is set out as
nucleotide sequence SEQ ID NO: 619 and encodes the amino acid
sequence set out as SEQ ID NO: 620. NTHi purE gene is set out as
nucleotide sequence SEQ ID NO: 621 and encodes the amino acid
sequence set out as SEQ ID NO: 622. NTHi ribB gene is set out as
nucleotide sequence SEQ ID NO: 623 and encodes the amino acid
sequence set out as SEQ ID NO: 624. NTHi arcB gene is set out as
nucleotide sequence SEQ ID NO: 625 and encodes the amino acid
sequence set out as SEQ ID NO: 626. NTHi uxuA gene is set out as
nucleotide sequence SEQ ID NO: 627 and encodes the amino acid
sequence set out as SEQ ID NO: 628. NTHi dsbB gene is set out as
nucleotide sequence SEQ ID NO: 629 and encodes the amino acid
sequence set out as SEQ ID NO: 630. NTHi ureH gene is set out as
nucleotide sequence SEQ ID NO: 631 and encodes the amino acid
sequence set out as SEQ ID NO: 632. NTHi licC gene is set out as
nucleotide sequence SEQ ID NO: 633 and encodes the amino acid
sequence set out as SEQ ID NO: 634. NTHi HI1647 gene is set out as
nucleotide sequence SEQ ID NO: 635 and encodes the amino acid
sequence set out as SEQ ID NO: 636. NTHi ispZ gene is set out as
nucleotide sequence SEQ ID NO: 637 and encodes the amino acid
sequence set out as SEQ ID NO: 638. NTHi radC gene is set out as
nucleotide sequence SEQ ID NO: 639 and encodes the amino acid
sequence set out as SEQ ID NO: 640. NTHi mukF gene is set out as
nucleotide sequence SEQ ID NO: 641 and encodes the amino acid
sequence set out as SEQ ID NO: 642. NTHi glpR gene is set out as
nucleotide sequence SEQ ID NO: 643 and encodes the amino acid
sequence set out as SEQ ID NO: 644. NTHi ihfB gene is set out as
nucleotide sequence SEQ ID NO: 645 and encodes the amino acid
sequence set out as SEQ ID NO: 646. NTHi argR gene is set out as
nucleotide sequence SEQ ID NO: 647 and encodes the amino acid
sequence set out as SEQ ID NO: 648. NTHi cspD gene is set out as
nucleotide sequence SEQ ID NO: 649 and encodes the amino acid
sequence set out as SEQ ID NO: 650. NTHi HI1163 gene is set out as
nucleotide sequence SEQ ID NO: 651 and encodes the amino acid
sequence set out as SEQ ID NO: 652. NTHi HI1063 gene is set out as
nucleotide sequence SEQ ID NO: 653 and encodes the amino acid
sequence set out as SEQ ID NO: 654. NTHi HI0665 gene is set out as
nucleotide sequence SEQ ID NO: 655 and encodes the amino acid
sequence set out as SEQ ID NO: 656. NTHi HI1292 gene is set out as
nucleotide sequence SEQ ID NO: 657 and encodes the amino acid
sequence set out as SEQ ID NO: 658.
[0010] The novel NTHi genes included in the polynucleotide
sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and
the nucleotide sequences set out in Tables 4 and 4B are also
up-regulated during infection of the middle ear and/or the
nasopharynx, and therefore are contemplated to encode OM vaccine
candidates and/or targets of chemotherapy. In addition, the
following NTHi genes are contemplated to be virulence-associated
genes and therefore are contemplated to encode possible OM vaccine
candidates and/or targets of chemotherapy: HI1386, HI1462, HI1369,
lav, HI1598. NTHi HI1386 gene sequence is set out as SEQ ID NO: 659
and encodes the amino acid sequence set out as SEQ ID NO: 660. NTHi
HI1462 gene sequence is set out as SEQ ID NO: 661 and encodes the
amino acid sequence set out as SEQ ID NO: 662. NTHi HI1369 gene
sequence is set out as SEQ ID NO: 665 and encodes the amino acid
sequence set out as SEQ ID NO: 666. NTHi lav gene sequence is set
out as SEQ ID NO: 663 and encodes the amino acid sequence set out
as SEQ ID NO: 664. NTHi HI1598 gene sequence is set out as SEQ ID
NO: 669 and SEQ ID NO: 671 and encodes the amino acid sequence set
out as SEQ ID NO: 670 and SEQ ID NO: 672. Additional NTHi genes
associated with virulence include the polynucleotide sequences
presented as SEQ ID NO: 667 and SEQ ID NO: 673.
[0011] As a method of treating or preventing NTHi infection, the
present invention contemplates administering a molecule that
inhibits expression or the activity of the NTHi polypeptides, which
are upregulated or active during infection. In particular, the
invention contemplates methods of treating or preventing NTHi
infection comprising modulating NTHi protein expression by
administering an antisense oligonucleotide that specifically binds
to NTHi genes that are upregulated during NTHi infections, such
genes include hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA,
dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD,
HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. The invention also
contemplates methods of treating or preventing NTHi infection
comprising administering antibodies or small molecules that
modulate the activity of the proteins encoded by theses genes. The
novel NTHi genes included in the polynucleotide sequences presented
as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide
sequences set out in Tables 4 and 4B are also up-regulated during
infection of the middle ear and/or the nasopharynx and therefore
antisense oligonucleotides that specifically bind these
polynucleotide sequences are also contemplated.
Polynucleotides and Polypeptides of the Invention
[0012] The present invention provides for the sequences of the NTHi
strain 86-028NP genome. This genomic sequence is presented as a
series of contig sequences denoted herein as "contigs 1-576". Each
contig is assigned a sequence identification number that correlates
with its "contig number". Therefore, the contigs of the present
invention as set out as SEQ ID NOS: 1-576. These contig
polynucleotide sequences may be assembled into the complete genome
sequence of the NTHi strain 86-028NP using routine methods. Upon
completion of 8-fold sequence analysis of the NTHi strain 82-028NP
genome, the genomic sequence was assembled into 11 contigs which
are denoted herein as SEQ ID NOS: 675-685.
[0013] The present invention provides for the NTHi polynucleotide
sequences and open reading frames contained within the contigs of
SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences
set out in Table 3B, Table 4B and Table 5. The present invention
also provides for the polypeptide sequences encoded by the NTHi
polynucleotides of the present invention such as the amino acid
sequences set out in Table 3B, Table 4B and Table 5. The invention
provides for polynucleotides that hybridize under stringent
conditions to (a) the complement of the nucleotides sequence of SEQ
ID NOS: 1-576; SEQ ID NOS: 675-685 and the nucleotide sequences set
out in Table 3B, Table 4B and Table 5 herein (b) a polynucleotide
which is an allelic variant of any polynucleotides recited above;
(c) a polynucleotide which encodes a species homolog of any of the
proteins recited above; or (d) a polynucleotide that encodes a
polypeptide comprising a specific domain or truncation of the NTHi
polypeptides of the present invention.
[0014] The NTHi polynucleotides of the invention also include
nucleotide sequences that are substantially equivalent to the
polynucleotides recited above. Polynucleotides according to the
invention can have, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more
typically at least 90%, 91%, 92%, 93%, or 94% and even more
typically at least 95%, 96%, 97%, 98% or 99% sequence identity to
the NTHi polynucleotides recited above.
[0015] Included within the scope of the nucleic acid sequences of
the invention are nucleic acid sequence fragments that hybridize
under stringent conditions to the NTHi nucleotide sequences of SEQ
ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set
out in Table 3B, Table 4B and Table 5 herein, or compliments
thereof, which fragment is greater than about 5 nucleotides,
preferably 7 nucleotides, more preferably greater than 9
nucleotides and most preferably greater than 17 nucleotides.
Fragments of, e.g., 15, 17, or 20 nucleotides or more that are
selective for (i.e., specifically hybridize to any one of the
polynucleotides of the invention) are contemplated. Probes capable
of specifically hybridizing to a polynucleotide can differentiate
NTHi polynucleotide sequences of the invention from other
polynucleotide sequences in the same family of genes or can
differentiate NTHi genes from other bacterial genes, and are
preferably based on unique nucleotide sequences.
[0016] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of stringent conditions for hybridization and
washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at
65-68.degree. C. or 0.015 M sodium chloride, 0.0015M sodium
citrate, and 50% formamide at 42.degree. C. See Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring
Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent
conditions (such as higher temperature, lower ionic strength,
higher formamide, or other denaturing agent) may also be used,
however, the rate of hybridization will be affected. In instances
wherein hybridization of deoxyoligonucleotides is concerned,
additional exemplary stringent hybridization conditions include
washing in 6.times.SSC 0.05% sodium pyrophosphate at 37.degree. C.
(for 14-base oligos), 48.degree. C. (for 17-base oligos),
55.degree. C. (for 20-base oligos), and 60.degree. C. (for 23-base
oligos).
[0017] Other agents may be included in the hybridization and
washing buffers for the purpose of reducing non-specific and/or
background hybridization. Examples are 0.1% bovine serum albumin,
0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium
dodecylsulfate, NaDodSO.sub.4, (SDS), ficoll, Denhardt's solution,
sonicated salmon sperm DNA (or other non-complementary DNA), and
dextran sulfate, although other suitable agents can also be used.
The concentration and types of these additives can be changed
without substantially affecting the stringency of the hybridization
conditions. Hybridization experiments are usually carried out at pH
6.8-7.4, however, at typical ionic strength conditions, the rate of
hybridization is nearly independent of pH. See Anderson et al.,
Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press
Limited (Oxford, England). Hybridization conditions can be adjusted
by one skilled in the art in order to accommodate these variables
and allow DNAs of different sequence relatedness to form
hybrids.
[0018] The sequences falling within the scope of the present
invention are not limited to these specific sequences, but also
include allelic and species variations thereof. Allelic and species
variations can be routinely determined by comparing the sequence
provided in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, and nucleotide
sequences out in Table 3B, Table 4B and Table 5 herein, preferably
the open reading frames therein, a representative fragment thereof,
or a nucleotide sequence at least 90% identical, preferably 95%
identical, to the open reading frames within SEQ ID NOS: 1-576, SEQ
ID NOS: 675-685 and the nucleotide sequences set out in Table 3B,
Table 4B and Table 5 with a sequence from another isolate of the
same species. Preferred computer program methods to determine
identity and similarity between two sequences include, but are not
limited to, the GCG program package, including GAP (Devereux et
al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group,
University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA
(Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX
program is publicly available from the National Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual,
Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al.,
supra). The well known Smith Waterman algorithm may also be used to
determine identity.
[0019] Furthermore, to accommodate codon variability, the invention
includes nucleic acid molecules coding for the same amino acid
sequences as do the specific open reading frames (ORF) disclosed
herein. In other words, in the coding region of an ORF,
substitution of one codon for another codon that encodes the same
amino acid is expressly contemplated.
[0020] The isolated polypeptides of the invention include, but are
not limited to, a polypeptide comprising: the amino acid sequences
encoded by the nucleotide sequences included within the
polynucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS:
675-685 and the nucleotide sequences set out in Table 3B, Table 4B
and Table 5, or the corresponding full length or mature protein.
The polypeptides of the invention include the amino acid sequences
of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622,
SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ
ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID
NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO:
646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO:
654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO:
662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO:
670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO:
689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO:
697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO:
705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO:
713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721,
SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID
NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO:
739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO:
747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753, SEQ ID NO:
755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID
NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID NO: 771 which are
set out in Table 3B, Table 4B and Table 5 herein.
[0021] Polypeptides of the invention also include polypeptides
preferably with biological or immunogenic activity that are encoded
by: (a) an open reading frame contained within the nucleotide
sequences set forth as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and
the nucleotide sequences set out in Table 3B, Table 4B and Table 5,
or (b) polynucleotides that hybridize to the complement of the
polynucleotides of (a) under stringent hybridization
conditions.
[0022] The invention also provides biologically active or
immunologically active variants of the amino acid sequences of the
present invention; and "substantial equivalents" thereof (e.g.,
with at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least
about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%,
97%, more typically at least about 98%, or most typically at least
about 99% amino acid identity) that retain biological and/or
immunogenic activity. Polypeptides encoded by allelic variants may
have a similar, increased, or decreased activity compared to
polypeptides encoded by the polynucleotides included within the
nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS:
675-685 and the nucleotide sequences set out in Table 3B, Table 4B
and Table 5 herein, and the polypeptides having an amino acid
sequence set out in Table 3B, Table 4B and Table 5 herein
[0023] NTHi peptides refer to fragments of the NTHi polypeptides
encoded by the nucleotide sequences presented in SEQ ID NOS: 1-576,
SEQ ID NOS: 675-685 or the nucleotide sequences set out in Table
3B, Table 4B and Table 5 herein, and the polypeptides having the
amino acid sequences set out in Table 3B, Table 4B and Table 5
herein. The preferred NTHi peptides are biologically and/or
immunologically active.
[0024] The present invention further provides isolated NTHi
polypeptides or NTHi peptides encoded by the NTHi nucleic acid
fragments of the present invention or by degenerate variants of the
nucleic acid fragments of the present invention. The term
"degenerate variant" refers to nucleotide fragments which differ
from a nucleic acid fragment of the present invention (e.g., an
ORF) by nucleotide sequence but, due to the degeneracy of the
genetic code, encode an identical NTHi polypeptide sequence.
Preferred nucleic acid fragments of the present invention are the
ORFs that encode proteins.
[0025] The invention also provides for NTHi polypeptides with one
or more conservative amino acid substitutions that do not affect
the biological and/or immunogenic activity of the polypeptide.
Alternatively, the NTHi polypeptides of the invention are
contemplated to have conservative amino acids substitutions which
may or may not alter biological activity. The term "conservative
amino acid substitution" refers to a substitution of a native amino
acid residue with a nonnative residue, including naturally
occurring and nonnaturally occurring amino acids, such that there
is little or no effect on the polarity or charge of the amino acid
residue at that position. For example, a conservative substitution
results from the replacement of a non-polar residue in a
polypeptide with any other non-polar residue. Further, any native
residue in the polypeptide may also be substituted with alanine,
according to the methods of "alanine scanning mutagenesis".
Naturally occurring amino acids are characterized based on their
side chains as follows: basic: arginine, lysine, histidine; acidic:
glutamic acid, aspartic acid; uncharged polar: glutamine,
asparagine, serine, threonine, tyrosine; and non-polar:
phenylalanine, tryptophan, cysteine, glycine, alanine, valine,
proline, methionine, leucine, norleucine, isoleucine. General rules
for amino acid substitutions are set forth in Table 1 below.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Preferred
Residues Exemplary Substitutions Substitutions Ala Val, Leu, Ile
Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser
Gln Asn Asn Glu Asp Asn Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg
Ile Leu, Val, Met, Ala, Phe, Leu Leu Norleucine, Ile, Val, Met, Leu
Lys Arg, 1,4 Diaminobutyric Arg Met Leu, Phe, Ile Leu Phe Leu, Val,
Ile, Ala, Tyr Arg Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp
Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,
Ala, Leu
[0026] Antisense polynucleotides complementary to the
polynucleotides encoding the NTHi polypeptides are also
provided.
[0027] The invention contemplates that polynucleotides of the
invention may be inserted in a vector for amplification or
expression. For expression, the polynucleotides are operatively
linked to appropriate expression control sequence such as a
promoter and polyadenylation signal sequences. Further provided are
cells comprising polynucleotides of the invention. Exemplary
prokaryotic hosts include bacteria such as E. coli, Bacillus,
Streptomyces, Pseudomonas, Salmonella and Serratia.
[0028] The term "isolated" refers to a substance removed from, and
essentially free of, the other components of the environment in
which it naturally exists. For example, a polypeptide is separated
from other cellular proteins or a DNA is separated from other DNA
flanking it in a genome in which it naturally occurs.
Antibodies and Methods for Eliciting an Immune Response
[0029] The invention provides antibodies which bind to antigenic
epitopes unique to (i.e., are specific for) NTHi polypeptides. Also
provided are antibodies which bind to antigenic epitopes common
among multiple H. influenzae subtypes but unique with respect to
any other antigenic epitopes. The antibodies may be polyclonal
antibodies, monoclonal antibodies, antibody fragments which retain
their ability to bind their unique epitope (e.g., Fv, Fab and
F(ab)2 fragments), single chain antibodies and human or humanized
antibodies. Antibodies may be generated by techniques standard in
the art.
[0030] It is known in the art that antibodies to the capsular
polysaccharide of H. influenzae exhibit the ability to kill
bacteria in vitro assays. These antibodies are also known to
protect against challenge with H. influenzae in animal model
systems. These studies indicate antibody to the capsular
polysaccharides are likely to elicit a protective immune response
in humans. The present invention provides for antibodies specific
for the NTHi polypeptides of the present invention and fragments
thereof, which exhibit the ability to kill both H. influenzae
bacteria and to protect humans from NTHi infection. The present
invention also provides for antibodies specific for the NTHi
polypeptides of the invention which reduce the virulence, inhibit
adherence, inhibit cell division, and/or inhibit penetration into
the epithelium of H. influenzae bacteria or enhance phagocytosis of
the H. influenzae bacteria.
[0031] In vitro complement mediated bactericidal assay systems
(Musher et al., Infect. Immun. 39: 297-304, 1983; Anderson et al.,
J. Clin. Invest. 51: 31-38, 1972) may be used to measure the
bactericidal activity of anti-NTHi antibodies. Further data on the
ability of NTHi polypeptides and NTHi peptides to elicit a
protective antibody response may be generated by using animal
models of infection such as the chinchilla model system described
herein.
[0032] It is also possible to confer short-term protection to a
host by passive immunotherapy via the administration of pre-formed
antibody against an epitope of NTHi, such as antibodies against
NTHi OMP, LOS or noncapsular proteins. Thus, the contemplated
vaccine formulations can be used to produce antibodies for use in
passive immunotherapy. Human immunoglobulin is preferred in human
medicine because a heterologous immunoglobulin may provoke an
immune response to its foreign immunogenic components. Such passive
immunization could be used on an emergency basis for immediate
protection of unimmunized individuals exposed to special risks.
Alternatively, these antibodies can be used in the production of
anti-idiotypic antibody, which in turn can be used as an antigen to
stimulate an immune response against NTHi epitopes.
[0033] The invention contemplates methods of eliciting an immune
response to NTHi in an individual. These methods include immune
responses which kill the NTHi bacteria and immune responses which
block H. influenzae attachment to cells. In one embodiment, the
methods comprise a step of administering an immunogenic dose of a
composition comprising a NTHi protein or NTHi peptide of the
invention. In another embodiment, the methods comprise
administering an immunogenic dose of a composition comprising a
cell expressing a NTHi protein or NTHi peptide of the invention. In
yet another embodiment, the methods comprise administering an
immunogenic dose of a composition comprising a polynucleotide
encoding a NTHi protein or NTHi peptide of the invention. The
polynucleotide may be a naked polynucleotide not associated with
any other nucleic acid or may be in a vector such as a plasmid or
viral vector (e.g., adeno-associated virus vector or adenovirus
vector). Administration of the compositions may be by routes
standard in the art, for example, parenteral, intravenous, oral,
buccal, nasal, pulmonary, rectal, or vaginal. The methods may be
used in combination in a single individual. The methods may be used
prior or subsequent to NTHi infection of an individual.
[0034] An "immunological dose" is a dose which is adequate to
produce antibody and/or T cell immune response to protect said
individual from NTHi infection, particularly NTHi infection of the
middle ear and/or the nasopharynx or lower airway. Also provided
are methods whereby such immunological response slows bacterial
replication. A further aspect of the invention relates to an
immunological composition which, when introduced into an individual
capable or having induced within it an immunological response. The
immunological response may be used therapeutically or
prophylactically and may take the form of antibody immunity or
cellular immunity such as that arising from CTL or CD4+ T cells. A
NTHi protein or an antigenic peptide thereof may be fused with
co-protein which may not by itself produce antibodies, but is
capable of stabilizing the first protein and producing a fused
protein which will have immunogenic and protective properties. Thus
fused recombinant protein, preferably further comprises an
antigenic co-protein, such as Glutathione-S-transferase (GST) or
beta-galactosidase, relatively large co-proteins which solubilize
the protein and facilitate production and purification thereof.
Moreover, the co-protein may act as an adjuvant in the sense of
providing a generalized stimulation of the immune system. The
co-protein may be attached to either the amino or carboxy terminus
of the first protein. Provided by this invention are compositions,
particularly vaccine compositions, and methods comprising the NTHi
polypeptides encoded by the polynucleotide of the invention or
antigenic peptides thereof.
[0035] The invention correspondingly provides compositions suitable
for eliciting an immune response to NTHi infection, wherein the
antibodies elicited block binding of NTHi bacterium to the host's
cells. The compositions comprise NTHi proteins or NTHi peptides of
the invention, cells expressing the NTHi polypeptide, or
polynucleotides encoding the polypeptides. The compositions may
also comprise other ingredients such as carriers and adjuvants.
[0036] Genes that are up-regulated in NTHi infection of the middle
ear and/or the nasopharynx and genes that are associated with NTHi
virulence are described herein. The polypeptides and peptides
thereof which are encoded by these NTHi genes are contemplated to
be useful for eliciting an immune response for treating or
preventing disorders associated with NTHi infection, such as OM.
Some of the polypeptides encoded by these genes include: histidine
biosynthesis protein, lipoprotein B, peptide ABC transporter,
periplasmic SapA precursor, outer membrane lipoproteins carrier
protein precursor, ribose transport system permease protein,
phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE,
phosphoribosylaminoimidazole carboxylase catalytic subunit,
ornithine carbamolytransferase, mannonate dehydratase, disulfide
oxidoreductase, urease accessory protein, phosphocholine
cytidylytransferase, putative pyridoxine biosynthesis protein,
singlet oxygen resistance protein, intracellular septation protein,
DNA repair protein, MukF protein, glycerol-3-phosphate regulon
repressor, integration host factor beta subunit, arginine
repressor, cold shock like protein, stress response protein, LicA,
MukF, RadA and those hypothetical proteins encoded by HI0094,
HI1163, HI0665, HI1292, HI1064, HI186, HI0352 genes. NTHi OMPs, LOS
and noncapsular proteins are also contemplated to elicit an immune
response for prevention and treatment of disorders associated with
NTHi infection.
[0037] An "immunogenic dose" of a composition of the invention is
one that generates, after administration, a detectable humoral
and/or cellular immune response in comparison to the immune
response detectable before administration or in comparison to a
standard immune response before administration. The invention
contemplates that the immune response resulting from the methods
may be protective and/or therapeutic.
[0038] The invention includes methods of blocking binding of NTHi
bacteria to host cells in an individual. The methods comprise
administering antibodies or polypeptides of the invention that
block binding of NTHi cellular attachment. Alternatively,
administration of one or more small molecules that block binding of
NTHi cell attachment is contemplated. In vitro assays may be used
to demonstrate the ability of an antibody, polypeptide or small
molecule of the invention to block NTHi cell attachment.
[0039] Pharmaceutical compositions comprising antibodies of the
invention, polypeptides of the invention and/or small molecules of
the invention that block NTHi cellular attachment are provided. The
pharmaceutical compositions may consist of one of the foregoing
active ingredients alone, may comprise combinations of the
foregoing active ingredients or may comprise additional active
ingredients used to treat bacterial infections. The pharmaceutical
compositions may comprise one or more additional ingredients such
as pharmaceutically effective carriers. Dosage and frequency of the
administration of the pharmaceutical compositions are determined by
standard techniques and depend, for example, on the weight and age
of the individual, the route of administration, and the severity of
symptoms. Administration of the pharmaceutical compositions may be
by routes standard in the art, for example, parenteral,
intravenous, oral, buccal, nasal, pulmonary, rectal, or
vaginal.
[0040] Also provided by the invention are methods for detecting
NTHi infection in an individual. In one embodiment, the methods
comprise detecting NTHi polynucleotides of the invention in a
sample using primers or probes that specifically bind to the
polynucleotides. Detection of the polynucleotide may be
accomplished by numerous techniques routine in the art involving,
for example, hybridization and PCR.
[0041] The antibodies of the present invention may also be used to
provide reagents for use in diagnostic assays for the detection of
NTHi antigens (NTHi polypeptides and peptides thereof) in various
body fluids of individuals suspected of H. influenzae infection. In
another embodiment, the NTHi proteins and peptides of the present
invention may be used as antigens in immunoassays for the detection
of NTHi in various patient tissues and body fluids including, but
not limited to: blood, serum, ear fluid, spinal fluid, sputum,
urine, lymphatic fluid and cerebrospinal fluid. The antigens of the
present invention may be used in any immunoassay system known in
the alt including, but not limited to: radioimmunoassays, ELISA
assays, sandwich assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
fluorescent immunoassays, protein A immunoassays and
immunoelectrophoresis assays.
Vaccines and Chemotherapeutic Targets
[0042] An aspect of the invention relates to a method for inducing
an immunological response in an individual, particularly a mammal
which comprises inoculating the individual with a NTHi antigen
protein or an antigenic peptide thereof.
[0043] The present invention also provides for vaccine formulations
which comprise an immunogenic recombinant NTHi protein or NTHi
peptide of the invention together with a suitable carrier. The NTHi
polypeptides and peptides thereof contemplated as vaccine
candidates and/or targets of chemotherapy include, but are not
limited to, histidine biosynthesis protein, lipoprotein B, peptide
ABC transporter, periplasmic SapA precursor, outer membrane
lipoproteins carrier protein precursor, ribose transport system
permease protein, phosphoribosylaminoimidazole carboxylase
catalytic subunit, PurE, 3,4-dihydroxt-2-butone 4-phosphate
synthase, ornithine carbamolytransferase, mannonate dehydratase,
disulfide oxidoreductase, urease accessory protein, phospshocholine
cytidylytransferase, putative pyridoxine biosynthesis protein,
singlet oxygen resistance protein, intracellular septation protein,
DNA repair protein, MUKF protein, glycerol-3-phosphate regulon
repressor, integration host factor beta subunit, arginine
repressor, cold shock like protein, stress response protein, LicA,
RadA and those hypothetical proteins encoded by HI0094, HI1163,
HI0665, HI1292, HI1064, HI1386, HI10352 genes, NTHi OMPs, NTHi LOS
and NTHi noncapsular proteins and polypeptides encoded by the novel
NTHi polynucleotide sequences present in the nucleotide sequences
set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the
nucleotide sequences set out in Table 3B, Table 4B and Table 5
herein, and the polypeptides having the amino acid sequences set
out in Table 3B, Table 4B and Table 5 herein.
[0044] Since the protein may be broken down in the stomach, it is
preferably administered parenterally, including, for example,
administration that is subcutaneous, intramuscular, intravenous, or
intradermal. Formulations suitable for parenteral administration
include aqueous and non-aqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the bodily fluid, preferably
the blood, of the individual; and aqueous and non-aqueous sterile
suspensions which may include suspending agents or thickening
agents. The formulations may be presented in unit-dose or
multi-dose containers, for example, sealed ampules and vials and
may be stored in a freeze-dried condition requiring only the
addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation may also include adjuvant systems for
enhancing the immunogenicity of the formulation, such as oil-in
water systems and other systems known in the art. The dosage will
depend on the specific activity of the vaccine and can be readily
determined by routine experimentation.
[0045] A. Peptide Vaccines
[0046] Peptide therapeutic agents, such as peptide vaccines, are
well known in the art and are of increasing use in the
pharmaceutical arts. Consistent drawbacks to the parenteral
administration of such peptide compounds have been the rapidity of
breakdown or denaturation. Infusion pumps, as well as wax or oil
implants, have been employed for chronic administration of
therapeutic agents in an effort to both prolong the presence of
peptide-like therapeutic agents and preserve the integrity of such
agents. Furthermore, the peptide-like agent should (with particular
reference to each epitope of the peptide-like agent) ideally
maintain native state configuration for an extended period of time
and additionally be presented in a fashion suitable for triggering
an immunogenic response in the challenged animal or immunized
human.
[0047] The NTHi antigenic peptides of the invention can be prepared
in a number of conventional ways. The short peptides sequences can
be prepared by chemical synthesis using standard means.
Particularly convenient are solid phase techniques (see, e.g.,
Erikson et al., The Proteins (1976) v. 2, Academic Press, New York,
p. 255). Automated solid phase synthesizers are commercially
available. In addition, modifications in the sequence are easily
made by substitution, addition or omission of appropriate residues.
For example, a cysteine residue may be added at the carboxy
terminus to provide a sulfhydryl group for convenient linkage to a
carrier protein, or spacer elements, such as an additional glycine
residue, may be incorporated into the sequence between the linking
amino acid at the C-terminus and the remainder of the peptide. The
short NTHi peptides can also be produced by recombinant techniques.
The coding sequence for peptides of this length can easily be
synthesized by chemical techniques, e.g., the phosphotriester
method described in Matteucci et al., J Am Chem Soc., 103: 3185
(1981).
[0048] Some of the NTHi peptide sequences contemplated herein may
be considered too small to be immunogenic, they may be linked to
carrier substances in order to confer this property upon them. Any
method of creating such linkages known in the art may be used.
Linkages can be formed with heterobifunctional agents that generate
a disulfide link at one functional group end and a peptide link at
the other, such as a disulfide amide forming agent, e.g.,
N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (See, e.g.,
Jansen et al., Immun. Rev. 62:185, 1982) and bifunctional coupling
agents that form a thioether rather than a disulfide linkage such
as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid,
2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic
acid and the like, and coupling agent which activate carboxyl
groups by combining them with succinimide or
1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as
succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate
(SMCC).
[0049] B. Vaccine Compositions and Administration
[0050] A priming dose of the immunogen that is followed by one or
more booster exposures to the immunogen may be necessary to be an
effective vaccine (Kramp et al., Infect. Immun., 25: 771-773, 1979;
Davis et al., Immunology Letters, 14: 341-8 1986 1987). Examples of
proteins or polypeptides that could beneficially enhance the immune
response if co-administered include cytokines (e.g., IL-2, IL-12,
GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory
molecules. Helper (HTL) epitopes could be joined to intracellular
targeting signals and expressed separately from the CTL epitopes.
This would allow direction of the HTL epitopes to a cell
compartment different than the CTL epitopes. If required, this
could facilitate more efficient entry of HTL epitopes into the MHC
class II pathway, thereby improving CTL induction. In contrast to
CTL induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0051] Ideally, an immunogen will exhibit two properties; the
capacity to stimulate the formation of the corresponding antibodies
and the propensity to react specifically with these antibodies.
Immunogens bear one or more epitopes which are the smallest part of
an immunogen recognizable by the combing site of an antibody. In
particular instances, immunogen, fractions of immunogens or
conditions under which the immunogen is presented are inadequate to
precipitate the desired immunological response resulting in
insufficient immunity. This is often the case with peptides or
other small molecules used as immunogens. Other substances such as
immunomodulators (e.g., cytokines such as the interleukins) may be
combined in vaccines as well.
[0052] The vaccine art recognizes the use of certain substances
called adjuvants to potentate an immune response when used in
conjunction with an immunogen. Adjuvants are further used to elicit
an immune response that is faster or greater than would be elicited
without the use of the adjuvant. In addition, adjuvants may be used
to create an immunological response using less immunogen than would
be needed without the inclusion of adjuvant, to increase production
of certain antibody subclasses that afford immunological protection
or to enhance components of the immune response (e.g., humoral,
cellular). Known adjuvants include emulsions such as Freund's
Adjuvants and other oil emulsions, Bordetella pertussis, MF59,
purified saponin from Quillaja saponaria (QS21), aluminum salts
such as hydroxide, phosphate and alum, calcium phosphate, (and
other metal salts), gels such as aluminum hydroxide salts,
mycobacterial products including muramyl dipeptides, solid
materials, particles such as liposomes and virosomes. Examples of
natural and bacterial products known to be used as adjuvants
include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like
acylated monosaccharide), OM-174 which is a lipid A derivative from
E. coli, holotoxins such as cholera toxin (CT) or one of its
derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of E.
coli or one of its derivatives, and CpG oligonucleotides. Adjuvant
activity can be affected by a number of factors, such as carrier
effect, depot formation, altered lymphocyte recirculation,
stimulation of T-lymphocytes, direct stimulation of B-lymphocytes
and stimulation of macrophages.
[0053] Vaccines are typically prepared as injectables, either as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid prior to injection may also be
prepared. The preparation may also be emulsified. The active
immunogenic ingredient is often mixed with excipients, which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, e.g., water, saline, dextrose,
glycerol, ethanol, or the like and combinations thereof. In
addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, or adjuvants, which enhance the effectiveness of
the vaccine. The vaccines are conventionally administered
parenterally, by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkalene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1-2%. Oral formulations include such normally employed
excipients as, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate and the like. These compositions take the form
of solutions, suspensions, tablets, pills, capsules, sustained
release formulations or powders and contain 10%-95% of active
ingredient, preferably 25-70%.
[0054] Vaccines may also be administered through transdermal routes
utilizing jet injectors, microneedles, electroporation,
sonoporation, microencapsulation, polymers or liposomes,
transmucosal routes and intranasal routes using nebulizers,
aerosols and nasal sprays. Microencapsulation using natural or
synthetic polymers such as starch, alginate and chitosan, D-poly
L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres,
polycaprolactones, polyorthoesters, polyanhydrides and
polyphosphazenes polyphosphatazanes are useful for both transdermal
and transmucosal administration. Polymeric complexes comprising
synthetic poly-ornithate, poly-lysine and poly-arginine or
amphipathic peptides are useful for transdermal delivery systems.
In addition, due to their amphipathic nature, liposomes are
contemplated for transdermal, transmucosal and intranasal vaccine
delivery systems. Common lipids used for vaccine delivery include
N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methyl
sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride
DOTMA, dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE),
dimethyldioctadecyl ammonium bromide (DDAB) and
9N(N',N-dimethylaminoethane)carbamoyl) cholesterol (DC-Chol). The
combination of helper lipids and liposomes will enhance up-take of
the liposomes through the skin. These helper lipids include,
diolcoyl phosplhatidylethanolamine (DOPE),
dilauroylphosphatidylethanolamine (DLPE), dimyristoyl
phosphatidylethanolamine (DMPE),
dipalmitoylphosphatidylethanolamine (DPPE). In addition,
triterpenoid glycosides or saponins derived from the Chilean soap
tree bark (Quillaja saponaria) and chitosan (deacetylated chitan)
have been contemplated as useful adjuvants for intranasal and
transmucosal vaccine delivery.
[0055] The proteins may be formulated into the vaccine as neutral
or salt forms. Pharmaceutically acceptable salts, include the acid
addition salts (formed with the free amino groups of the peptide)
and which are formed with inorganic acids such as, e.g.,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic. Salts formed with the free carboxyl
groups may also be derived from inorganic bases such as, e.g.,
sodium, potassium, ammonium, calcium, or ferric hydroxides, and
such organic bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, and procaine.
[0056] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, capacity of the
subject's immune system to synthesize antibodies, and the degree of
protection desired. Precise amounts of active ingredient required
to be administered depend on the judgment of the practitioner and
are peculiar to each individual. However, suitable dosage ranges
are of the order of several hundred micrograms active ingredient
per individual. Suitable regimes for initial administration and
booster shots are also variable, but are typified by an initial
administration followed in one or three month intervals by a
subsequent injection or other administration.
[0057] Upon immunization with a vaccine composition as described
herein, the immune system of the host responds to the vaccine by
producing large amounts of CTLs specific for the desired antigen,
and the host becomes at least partially immune to later infection,
or resistant to developing chronic infection. Vaccine compositions
containing the NTHi polypeptide or NTHi peptides of the invention
are administered to a patient susceptible to or otherwise at risk
of bacterial infection to elicit an immune response against the
antigen and thus enhance the patient's own immune response
capabilities. Such an amount is defined to be an "immunogenically
effective dose." In this use, the precise amounts again depend on
the patient's state of health and weight, the mode of
administration, the nature of the formulation, etc., but generally
range from about 1.0 .mu.g to about 5000 per 70 kilogram patient,
more commonly from about 10 to about 500 mg per 70 kg of body
weight. For therapeutic or immunization purposes, the NTHi
polypeptide or NTHi peptides of the invention can also be expressed
by attenuated viral hosts, such as vaccinia or fowlpox. This
approach involves the use of vaccinia virus as a vector to express
nucleotide sequences that encode the peptides of the invention.
Upon introduction into an acutely or chronically infected host or
into a noninfected host, the recombinant vaccinia virus expresses
the immunogenic peptide, and thereby elicits a host CTL
response.
[0058] Humoral immune response may be measured by many well known
methods, such as Single Radial Inmunodiffussion Assay (SRID),
Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay
(HAI). In particular, SRID utilizes a layer of a gel, such as
agarose, containing the immunogen being tested. A well is cut in
the gel and the serum being tested is placed in the well.
[0059] Diffusion of the antibody out into the gel leads to the
formation of a precipitation ring whose area is proportional to the
concentration of the antibody in the serum being tested. EIA, also
known as ELISA (Enzyme Linked Immunoassay), is used to determine
total antibodies in the sample. The immunogen is adsorbed to the
surface of a microtiter plate. The test serum is exposed to the
plate followed by an enzyme linked immunoglobulin, such as IgG. The
enzyme activity adherent to the plate is quantified by any
convenient means such as spectrophotometry and is proportional to
the concentration of antibody directed against the immunogen
present in the test sample. HAI utilizes the capability of an
immunogen such as viral proteins to agglutinate chicken red blood
cells (or the like). The assay detects neutralizing antibodies,
i.e., those antibodies able to inhibit hemagglutination. Dilution
of the test serum are incubated with a standard concentration of
immunogen, followed by the addition of the red blood cells. The
presence of neutralizing antibodies will inhibit the agglutination
of the red blood cells by the immunogen. Tests to measure cellular
immune response include determination of delayed-type
hypersensitivity or measuring the proliferative response of
lymphocytes to target immunogen.
[0060] Nontypeable Haemophilus influenzae (NTHi) p H. influenzae is
a small, nonmotile gram negative bacterium. Unlike other H.
influenzae strains, the nontypeable H. influenzae (NTHi) strains
lack a polysaccharide capsule and are sometimes denoted as
"nonencapsulated." NTHi strains are genetically distinct from
encapsulated strains and are more heterogenous than the type b H.
influenzae isolates. NTHi presents a complex array of antigens to
the human host. Possible antigens that may elicit protection
include OMPs, lipopolysaccharides, lipoproteins, adhesion proteins
and noncapsular proteins.
[0061] Humans are the only host for H. influenzae. NTHi strains
commonly reside in the upper respiratory tract including the
nasopharynx and the posterior oropharynx, the lower respiratory
tract and the female genital tract. NTHi causes a broad spectrum of
diseases in humans, including but not limited to, otitis media,
pneumonia, sinusitis, septicemia, endocarditis, epiglottitis,
septic arthritis, meningitis, postpartum and neonatal infections,
postpartum and neonatal sepsis, acute and chromic salpingitis,
epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis,
cholecystitis, intraabdominal infections, urinary tract infection,
mastoiditis, aortic graft infection, conjunctitivitis, Brazilian
purpuric fever, occult bacteremia and exacerbation of underlying
lung diseases such as chronic bronchitis, bronchietasis and cystic
fibrosis.
[0062] Epidemiologic studies of NTHi have indicated that the
strains are heterogeneous with respect to outer membrane protein
profiles (Barenkamp et al., Infect. Immun., 36: 535-40, 1982),
enzyme allotypes (Musser et al., Infect. Immun., 52: 183-191,
1986), and other commonly used epidemiologic tools. There have been
several attempts to subtype NTHi, but none of the methodologies
have been totally satisfactory. The outer-membrane protein
composition of NTHi consists of approximately 20 proteins. All NTHi
strains contains two common OMP's with molecular weights of 30,000
and 16,600 daltons. NTHi strains may be subtyped based on two OMP's
within the 32,000-42,000 dalton range. The NTHi liposaccharide
profile is fundamentally different than the enteric gram negative
bacteria and separates into 1-4 distinct bands ranging from less
than 20,000 daltons.
[0063] A prototype NTHi isolate is the low passage isolate 86-028NP
which was recovered from a child with chronic otitis media. This
strain has been well characterized in vitro (Bakaletz et al.,
Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog.,
23: 157-66, 1997) as well as in the chinchilla OM model (described
herein) (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzuki et al.,
Infect. Immun., 62: 1710-8, 1994; DeMaria et al., Infect. Immun.,
64: 5187-92, 1996). The 86-028NP strain was used, as described
herein, to identify genes that are up-regulated in expression in
the chinchilla model of otitis media and genes that are necessary
for NTHi survival in the chinchilla middle ear.
DFI Strategy
[0064] A differential fluorescence induction (DFI) strategy was
used herein to identify NTHi genes induced during OM in a
chinchilla animal model. Several methods have been developed to
identify bacterial genes that contribute to the virulence of an
organism during infection. Such methods include in vivo expression
technology (IVET) in which bacterial promoters regulate the
expression of gene(s) required for synthesis of essential nutrients
required for survival in the host; signature-tagged mutagenesis
(STM) enabling tag-specific identification of genes that alter the
virulence properties of a microorganism when mutated; DNA
microarray technology to globally screen for transcriptionally
active genes, and DFI which uses FACS analysis to select for
transcriptionally active promoters (Chiang et al., Annu. Rev.
Microbiol., 53: 129-154, 1999). DFI is a high-throughput method
that allows for the identification of differentially regulated
genes regardless of the basal level of expression and does not
exclude those that are essential for growth in vitro.
[0065] DFI has been successfully utilized in many microorganisms.
For example, a GFP reporter system and flow cytometry was used to
study mycobacterial gene expression upon interaction with
macrophages (Dhandayuthapani et al., Mol. Microbiol., 17: 901-912,
1995). A promoter trap system was used to identify genes whose
transcription was increased when Salmonellae were subjected to
environments simulating in vitro growth and when internalized by
cultured macrophage-like cells (Valdivia and Falkow, Mol.
Microbiol., 22: 367-378, 1996; Valdivia and Falkow, Science, 277:
2007-2011, 1997; Valdivia and Falkow, Curr. Opin. Microbiol., 1:
359-363, 1998). In addition, DFI has been used to identify
promoters expressed in S. pneumoniae and S. aureus when grown under
varied in vitro conditions simulating infection (Marra et al.,
Infect. Immun., 148: 1483-1491, 2002; Schneider et al., Proc. Natl.
Acad. Sci. U.S.A., 97: 1671-1676, 2000). In addition, DFI has been
utilized to study gene regulation in Bacillus cereus in response to
environmental stimuli (Dunn and Handelsman, Gene, 226: 297-305,
1999), in S. pneumoniae in response to a competence stimulatory
peptide (Bartilson et al., Mol. Microbiol., 39: 126-135, 2001), and
upon interaction with and invasion of host cells in Bartonella
henselae Lee and Falkow, Infect. Immun., 66: 3964-3967, 1998),
Listeria monocytogenes Wilson et al., Infect. Immun., 69:
5016-5024, 2001), Brucella abortus (Eskra et al., Infect. Immun.,
69: 7736-7742, 2001), and Escherichia coli (Badger et al., Mol.
Microbiol., 36: 174-182, 2000).
[0066] Whereas DFI has been successfully used to identify promoters
active in cell culture models of infection or in vitro conditions
designed to simulate an in vivo environment, few have applied DFI
to identify promoters regulated in a specific biological niche
within the whole animal. This is likely due to the numerous
challenges associated with sorting from an in vivo environment. The
host inflammatory response, dissemination and/or clearance of
bacterial cells from the site of infection, as well as adherence of
bacteria to epithelial cells, possibly via biofilm formation, can
make bacteria inaccessible for retrieval from the living animal.
These factors, among others, contribute to the complexity of the
microenvironment and the heterogeneity of gene expression as the
bacteria sense and respond to these changes. Recently, DFI has been
used to identify promoters expressed in S. pneumoniae when the
bacteria were screened in a mouse model of respiratory tract
infection and a gerbil infection model of OM (Marra et al., Infect.
Immun. 70: 1422-33, 2002; Marra et al., Microbiol., 148: 1483-91,
2002).
Animal Model
[0067] The chinchilla model is a widely accepted experimental model
for OM. In particular, a chinchilla model of NTHi-induced OM has
been well characterized (Bakaletz et al., J. Infect. Dis., 168:
865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol., 4:
223-225, 1997; Suzuki and Bakaletz, Infest. Immun., 62: 1710-1718,
1994), and has been used to determine the protective efficacy of
several NTHi outer membrane proteins, combinations of outer
membrane proteins, chimeric synthetic peptide vaccine components,
and adjuvant formulations as vaccinogens against OM (Bakaletz et
al., Vaccine, 15; 955-961, 1997; Bakaletz et al., Infect. Immun.,
67: 2746-2762, 1999; Kennedy et al., Infect. Immun., 68: 2756-2765,
2000).
[0068] In particular, there is an unique in vivo model wherein
adenovirus predisposes chinchillas to H. influenzae-induced otitis
media, which allowed for the establishment of relevant cell, tissue
and organ culture systems for the biological assessment of NTHi
(Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993; Suzuki et
al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infection alone
has been used to assess for the transudation of induced serum
antibodies into the tympanum (Bakaletz et al., Clin. Diagnostic Lab
Immunol., 4(2): 223-5, 1997) and has been used as a co-pathogen
with NTHi, to determine the protective efficacy of several active
and passive immunization regimens targeting various NTHi outer
membrane proteins, combinations of OMPs, chimeric synthetic peptide
vaccine components, and adjuvant formulations as vaccinogens
against otitis media (Bakaletz et al., Infect Immunity, 67(6):
2746-62, 1999; Kennedy et al., Infect Immun., 68(5): 2756-65, 2000;
Novotny et al., Infect Immunity 68(4): 2119-28, 2000; Poolman et
al., Vaccine 19 (Suppl. 1): S108-15, 2000).
Genes Upregulated In Vivo in Response to NTHi Infection of the
Middle Ear
[0069] In order to identify differentially regulated promoters in
response to NTHi infection of the middle ear, a promoter trap
library was constructed and sorting parameters defined. A portion
of the promoter trap library was inoculated directly into the
chinchilla middle ear and OM development was monitored by video
otoscopy and tympanometry at 24 and 48 hours. In addition, the
middle ear fluids were recovered 24 and 48 hours after infection.
Two-color FACS analysis was used to isolated bacteria that were
expressing GFP from other cells and debris associated with the
effusion. Following isolation, DNA sequence of the Haemophilus
inserts 5' of the gfpmut3 gene were determined and analyzed. In
this manner, we identified genes that are up-regulated as NTHi
sense and respond to the environment of the chinchilla middle ear
during AOM. The following genes were identified and due to their
up-regulation during NTHi infection, they may play a role in NTHi
infection and virulence.
[0070] As described below in Example 7, following the DFI procedure
described above and subsequent FACS analysis of gfp-expressing
clones, 52 candidate clones containing potential in vivo-regulated
promoters were isolated. The genes these clones control were
categorized based upon general description and function within the
cell and include general metabolic processes, environmental
informational processing and membrane transport, membrane proteins
and hypothetical proteins. Eight of these 52 clones contain
sequences that are unique to NTHi strain 86-028NP. Importantly, 3
clones were isolated from independent screens in more than one
animal thereby verifying the method of isolation.
[0071] In order to independently confirm the FACS data, we
determined the relative expression of candidate genes by
quantitative RT-PCR. The parent strain 86-028NP, was used for these
studies. Thus, wild-type gene expression without the influence of
plasmid copy number on gene regulation was analyzed, allowing for
the indication of false-positive clone identification by FACS. Of
the 44 candidate clones containing sequence similar to that
identified in H. influenzae strain Rd, quantitative comparison of
gene expression in vitro and in vivo confirmed up-regulated gene
expression for twenty-six genes (60%) when NTHi respond to
environmental cues present in the chinchilla middle ear. This
analysis identified in vivo-regulated promoters which drive
expression of genes involved in membrane transport, environmental
informational processing, cellular metabolism, gene regulation, as
well as hypothetical proteins with unknown function. (See Table 4
in Example 6).
[0072] Quantitative RT-PCR demonstrated a two-fold increase in lolA
expression, enabling lipoprotein transport from the inner membrane
to the outer membrane. Bacteria grow rapidly in the middle ear
environment reaching 5.0.times.10.sup.8 CFU NTHi ml middle ear
fluid within 48 hours. The bacteria sense and respond to the
environment, acquiring or synthesizing the necessary nutrients for
growth and survival. The gene encoding the membrane component in
ribose sugar transport, rbsC (SEQ ID NO: 619), showed a 5-fold
increase in expression in vivo compared to cells growing in vitro.
In addition, many genes involved in metabolic processes show a
dramatic increase in gene expression in vivo compared to cells
growing in vitro. These include a riboflavin synthesis gene, ribB
(SEQ ID NO: 623), a purine nucleotide biosynthetic gene purE (SEQ
ID NO: 621), ornithine carbamoyltransferase, arcB (SEQ ID NO: 625),
involved in arginine degradation via the urea cycle and uxuA (SEQ
ID NO: 627), encoding mannonate hydrolase, required for the uptake
of D-glucuronate and transformation into glyceraldehyde
3-phosphate. In addition, but to a lesser degree, genes for
histidine biosynthesis (hisB; SEQ ID NO: 615), DNA repair (radC;
SEQ ID NO: 639) and a putative intracellular septation
transmembrane protein (ispZ; SEQ ID NO: 637) were up-regulated.
[0073] Disulfide bond formation is important for folding and
assembly of many secreted proteins in bacteria. In prokaryotes,
DsbA and DsbB make up the oxidative pathway responsible for the
formation of disulfides. DsbB reoxidizes DsbA, which donates
disulfide bonds directly to unfolded polypeptides, and DsbB has
been demonstrated to generate disulfides de novo from oxidized
quinones (Collet and Bardwell, Mol. Microbiol., 44: 1-8, 2002). In
H. influenzae strain Rd, DsbA is required for competence for
transformation (Tomb, Proc. Natl. Acad. Sci. U.S.A., 89:
10252-10256, 1992). Herein, an approximate 3-fold increase in dsbB
gene (SEQ ID NO: 629) transcription was demonstrated, illuminating
an important role for disulfide interchange for NTHi growing in the
middle ear environment.
[0074] Bacteria colonization of the middle ear, a normally sterile
environment, results in a host inflammatory response and subsequent
neutrophil infiltration. Bacteria have evolved numerous strategies
to combat this host response. NTHi increase gene expression
(4-fold) of ureH (SEQ ID NO:631), a homologue of a gene required
for expression of active urease in Helicobacter, shown to be
involved in acid tolerance (Young et al., J. Bacterol., 178:
6487-6495, 1996). Recently, it has been reported that urease
activity may play a role in chronic Actinobacillus pleuropneumoniae
infection by counteracting the decrease in pH occurring upon
infection (Baltes et al., Infect. Immun., 69: 472-478, 2000; Baltes
et al., Infect. Immun., 69: 472-478, 2001; Bosse and MacInnes, Can.
J. Vet. Res., 64: 145-150). A biotype analysis on NTHi isolates
from middle ear effusions demonstrated that 87% are urease positive
(DeMaria et al., J. Clin. Microbiol., 20: 1102-1104, 1984).
However, the role of urease in NTHi virulence is unknown.
Similarly, an increase in expression of a gene whose product
demonstrates 88% sequence identity to a pyridoxine biosynthesis
protein in S. pneumoniae and 60% homology to a putative singlet
oxygen resistance protein that may function as an antioxidant.
Phosphorylcholine (ChoP) has been implicated in the pathogenesis of
NTHi (Weiser et al., Infect. Immun., 65: 943-950, 1997). NTHi
modulates ChoP expression by phase variation, decorating the LOS on
the cell surface. ChoP may contribute to NTHi persistence in the
respiratory tract via decreased susceptibility to antimicrobial
peptides (Lysecko et al., Infect. Immun., 68: 1664-1671, 2000) and
alter the sensitivity to serum killing mediated by C-reactive
protein (CRP) (Weiser et al., J. Exp. Med., 187: 631-640, 1998).
The microenvironment of the nasopharynx and middle ear cavity may
select for the ChoP.sup.+ phenotype, as ChoP.sup.+ strains show
greater colonization of the chinchilla nasopharynx (Tong et al.,
Infect. Immun., 68: 4593-4597, 2000). Expression of the licC gene
(SEQ ID NO: 633) was also increased. The licC gene encodes a
phosphorylcholine cytidylyltransferase that plays a role in the
biosynthesis of phosphorylcholine-derivatized LOS (Rock et al., J.
Bacterol., 183: 4927-4931, 2001).
[0075] Also included among the in vivo-induced genes is a set whose
products subsequently regulate gene expression or DNA replication.
These genes include transcriptional regulation of glycerol
metabolism by the glp repressor, glpR (SEQ ID NO: 643), the
arginine repressor gene, argR (SEQ ID NO: 647), and the integration
host factor (IHF) beta subunit, ihfB (SEQ ID NO: 645). IHF is a
histone-like protein that binds DNA at specific sequences, an
accessory factor involved in replication, site-specific
recombination and transcription, altering the activity of a large
number of operons (Goosen and van de Putte, Mol. Microbiol. 16:
1-7, 1995). In addition, CspD inhibits DNA replication during
stationary phase-induced stress response in E. coli (Yamanaka et
al., Mol. Microbiol., 39: 1572-1584, 2001) and the mukF (SEQ ID NO:
641) gene protein homologue contributes to a remodeling of the
nucleiod structure into a more compact form prior to cell
segregation (Sawitzke and Austin, Proc. Natl. Acad. Sci. U.S.A.,
62: 1710-1718, 2000). The DFI strategy described herein also
identified promoters induced in vivo for genes of unknown function.
The hypothetical protein, HI0094, demonstrated an 8-fold increase
in gene expression during early OM but its role remains unknown.
HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the
hypothetical YdiJ proteins, a putative oxidase, of E. coli.
[0076] A high-density transposon mutagenesis strategy was used to
identify H. influenzae genes essential for growth on rich medium
(Akerley et al., Proc. Natl. Acad. Sci. U.S.A., 99: 966-971, 2002).
Six genes were identified in the screen described herein that are
included in essential gene set described in Akerley' et al., supra.
(hisB, lppB, lolA, ispZ, mukF and unknown HI0665). Recently genes
of non-typeable H. influenzae that are expressed upon interaction
with two human respiratory tract-derived epithelial cell lines have
been identified. These genes included those involved in metabolic
processes, stress responses, gene expression, cell envelope
biosynthesis, DNA-related processes, cell division and ORF's
encoding proteins of unknown function. (Ulsen et al., Mol.
Microbiol., 45:485-500, 2,002). Similarly the stress response gene,
cspD (SEQ ID NO: 649), genes involved in purine and riboflavin
biosynthesis, and a protein of unknown function, vapA was
identified in the screen described herein. Expression of vapA was
detected in vitro, yet vapA gene expression increased two-fold in
vivo. These unique approaches identified known genes that are
upregulated in NTHi-induced OM and therefore are likely to play a
role in NTHi infection and virulence; and may be potential
candidates for vaccines and antisense therapies and other
therapeutic methods of treatment of NTHi related disorders.
[0077] The DFI strategy resulted in the identification of promoters
induced in vivo for genes of unknown function as well. The
hypothetical protein, HI0094, demonstrated a 8-fold increase in
gene expression during early OM but its role remains unknown.
HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the
hypothetical YdiJ proteins, a putative oxidase, of E. coli.
Therefore, these hypothetical genes are likely to play a role in OM
induced by NTHi infection.
BRIEF DESCRIPTION OF FIGURES
[0078] FIG. 1 depicts the LKP gene region in a panel of Haemophilus
isolates. The strain 86-028NP sequence is identical in this region
to the sequence in NTHi strain R3001. Both of these NTHi lack the
hif gene cluster encoding the hemagglutinating pilus.
[0079] FIG. 2 depicts the rfaD region in a panel of Haemophilus
isolates. The gene arrangement in the rfaD region of the strain
86-028NP genome is similar to that of the strain Rd genome but
different than the arrangement of these genes seen in the genome of
most NTHi examined.
[0080] FIGS. 3A-3M set out the nucleotide sequences (SEQ ID NOS:
589-614) described in Table 4, which were identified to be
upregulated during OM infection (see Example 6). The nucleotides
(nt.) which correspond to known genes and those nt. which
correspond to the contig sequences set out as SEQ ID NO: 1-576 are
also presented.
DETAILED DESCRIPTION
[0081] The following examples illustrate the invention wherein
Example 1 describes the sequence of the NTHi genome, Example 2
describes the identified contigs and initial gene discovery,
Example 3 describes construction of the NTHi promoter trap library,
Example 4 describes the analyses of 86-028NP derivatives expressing
GFP, Example 5 demonstrates direct labelling of bacteria from
middle ear fluids, Example 6 describes identification of promoters
induced in vivo in acute otitis media, Example 7 describes
identification of virulence-associated genes, and Example 8
describes identification of unique NTHi gene sequences.
EXAMPLE 1
Sequence of a Non-Typeable Haemophilus influenzae Genome
[0082] NTHi strain 86-028NP is a minimally passaged clinical
isolate obtained from a pediatric patient who underwent
tympanostomy and tube insertion for chronic OM at Columbus
Children's Hospital. (Bakaletz et al. Infection and Immunity,
56(2): 331-335, 1988) The 86-028NP strain was deposited with the
American Type Tissue Collection (Manassas, Va. 20108 USA) on Oct.
16, 2002 and assigned accession no. PTA-4764.
[0083] In an effort to more broadly approach the identification of
the virulence determinants in NTHi, the genome of the NTHi 86-028NP
strain was sequenced to 3-fold coverage. Chromosomal DNA was
prepared from strain 86-028NP using the Puregene protocol and
sheared to 2-4 kb in size with a Hydroshear instrument (Gene
Machines). The sheared DNA was ethanol-precipitated, end-repaired
using a mixture of Klenow enzyme and T4 DNA polymerase, and
size-selected by agarose gel electrophoresis to obtain 2-4 kb
fragments as described in Chissoe et al. (Methods: a Companion to
Methods of Enzymology 3: 55-65, 1991) and Sambrook et al.
(Molecular Cloning: a Laboratory Manual, 2.sup.nd Ed. Cold Springs
Harbor, N.Y., 1989). These fragments were cloned into vector pUC18
using the SmaI restriction site (phosphatase-treated) and
transformed into E. coli XL-1 Blue, selecting for ampicillin
resistance. Colonies that contain inserts were identified by
blue/white screening on LB-Amp plates containing X-gal, and
transferred into 96-deep well plates containing 1.5 ml of TB-Amp
(TB=Terrific Broth) broth. The deep-well plate cultures were grown
overnight (18-22 hours) at 37.degree. C. Template preparation,
sequencing and contig assembly were performed.
[0084] Automated template preparation was performed on the Beckman
Biomek 2000 automated robotics workstation as described in Chissoe
et al., (supra.) Briefly, each 96-deep well plate, containing the
clones prepared above, was centrifuged to pellet the cells, the
supernatant decanted, and the cells frozen (if necessary) at
-20.degree. C. Four 96-deep well blocks were placed on the Biomek
table, and the liquid handling robot was used to prepare the
template using an automated version of a typical SDS-NaOH lysis
protocol as described in Chissoe et al., (supra.). The final
ethanol-precipitated templates were each dissolved in 50 .mu.l
ddH.sub.2O, and used for DNA sequencing.
[0085] Sequencing reactions were run by re-arraying the templates
(from 96-well plates) into 384-well plates, using the Robbins Hydra
96 robot. Cycle-sequencing reactions were run using PE Big-Dye.TM.
terminators and universal primers (M13 forward and reverse),
cleaned up over Sephadex G50 columns, and analyzed on a PE
Biosystems 3700 capillary electrophoresis DNA sequencer according
to the manufacturer's instructions. Sequencing reads (8219) were
assembled into 576 contigs (SEQ ID NOS: 1-576 herein). The
statistics for the 3-fold sequencing are shown in Table 2A. The
total unique sequence in assembly 17 is 1.74 Mb. TABLE-US-00002
TABLE 2A Contig Size Total Number Total Length % of Cumulative 0-1
kb 65 55961 3.2% 1-2 kb 228 333665 19.2% 2-3 kb 101 243059 14.0%
3-4 kb 49 172385 9.9% 4-5 kb 45 196699 11.3% 5-10 kb 74 515152
29.6% 10-20 kb 11 144591 8.3% 20-30 kb 3 77352 4.4%
[0086] Subsequently, 8-fold sequencing analysis of the NTHi genome
was carried out. The 8-fold sequencing assembled the NTHi genome
into 11 contigs. Contigs 5, 8, 9, 10, 12-18 are denoted as SEQ ID
NOS: 675-685 herein. The statistics for the 8-fold sequencing are
shown in Table 2B. TABLE-US-00003 TABLE 2B Contig Size Total Number
Total Length % of Cumulative 0-1 kb 5 3950 0.2% 1-2 kb 3 4316 0.2%
2-3 kb 0 0 0.0% 3-4 kb 1 3964 0.2% 4-5 kb 0 0 0.0% 5-10 kb 0 0 0.0%
10-20 kb 1 15147 0.8% 20-30 kb 2 51888 2.7% 30-40 kb 0 0 0.0% 40-50
kb 0 0 0.0% 50-100 kb 1 85814 4.5% >100 kb 5 1760339 91.4%
EXAMPLE 2
Contig Description and Initial Gene Discovery
[0087] Seventy-five of the 88 contigs with length.gtoreq.5000bp,
identified with the 3-fold sequence analysis, show significant
similarity via BLASTN to genes in H. influenzae strain Rd. To
visualize the potential relationship between the gene order in H.
influenzae strain 86-028NP and H. influenzae strain Rd, the
86-028NP three-fold contig set and the Rd gene set were
bidirectionally compared using BLASTN. The results were plotted in
gene-order verses contig space by sorting the contigs based on gene
coordinates of the Rd genes hit, anchoring each contig at the
smallest coordinate found as described in Ray et al.,
(Bioinformatics 17: 1105-12, 2001). Compared in this fashion, an
incomplete assembly of a genome with identical gene order to a
completely known genome would display a monotonically increasing
stair-stepped form.
[0088] BLASTX was used to identify hits to sequences with homology
to genes in the strain Rd genome as well as genes not found in H.
influenzae strain Rd. Hits to strain Rd sequences were removed from
the data set and the other hits summarized in Table 3A. The data
are presented as follows: contig # (=SEQ ID NO: #), column 1; E
score for each hit, column 2; the name of the protein that had
homology to a portion of the amino acid translation of the cited
contig, column 3; the organism producing the homologue, column 4;
and the Genbank protein identifier for each of the proteins cited
in column 3, column 5; the corresponding nucleotides within the
contig (referenced by SEQ ID NO:). In most instances, several
homologues were identified but for clarity, the protein of greatest
homology is cited in Table 3A.
[0089] The sequences for some of the genes listed in Table 3A were
identified within the 8-fold sequencing of the NTHi genome. Table
3B lists the location of these genes within the 11 contigs, the
full length open reading frame sequence (identified by SEQ ID NO:),
the derived amino acid sequence encoded by the open reading frame
and the gene with high homology identified by BLASTX (as listed in
Table 3A).
[0090] To examine the relative short range gene arrangements in
NTHi and the Rd strain, the gene order in two gene clusters that
have been well-described were compared. First, the genes present in
the hemagglutinating pilus (LKP) gene region were examined.
(Mhlanga-Mutangadura et al., J. Bacteriol. 180(17): 4693-703,
1998). The pilus gene cluster is located between the purE and pepN
genes, only fragments of which are depicted in FIG. 1. The serotype
b strain, Eagan, contains the hifABCDE gene cluster and produces
hemagglutinating pili. Strain Rd lacks the hicAB genes as well as
the hifABCDE gene cluster. In general, the nontypeable strains
previously examined contained the hicAB genes but not the hifgenes
that encode the hemagglutinating pilus. The strain 86-028NP
sequence (described herein) is identical in this region to the
sequence in NTHi strain R3001 (FIG. 1). The rfaD gene encodes an
enzyme involved in the biosynthesis of endotoxin. In addition, the
rfaD gene from NTHi strain 2019 has been characterized by Nichols
et al. (Infect Immunity 65(4): 1377-86, 1997). In strain 2019, the
rfaD gene is immediately upstream of the rfaF gene that encodes
another enzyme involved in endotoxin biosynthesis. The gene
arrangement in strain Rd is different; the rfaD and rfaF genes are
separated by approximately 11 kb of sequence. Most nontypeable
strains examined contained the gene arrangement seen in strain
2019. In contrast, strain 86-028NP has a gene arrangement identical
to that seen in strain Rd (FIG. 2).
[0091] A global analysis of the current assembly indicates that the
gene content and order are similar to that in strain Rd. A more
detailed analysis revealed that there are a substantial number of
NTHi genes not previously seen in the Pasteurellaceae and some
regions where the NTHi gene content and order is different than
that seen in strain Rd. Thus, the current data suggest that the
strain 86-028NP genome will contain a complex mosaic of Rd and
non-Rd like features.
[0092] The DFI strategy also identified novel NTHi sequences that
had increased gene expression. A list of these novel contig
sequences that contain genes or gene fragments that have homology
to ORFs in other organisms (primarily gram-negative bacteria) is
set out in Table 3A. For example, the nucleotide sequence of contig
442 (SEQ ID NO: 442), nucleotides 1498-1845 are highly homologous
to the sequences encoding amino acids 1-116 of H. influenzae strain
Rd lipoprotein B (LppB). The gene is positioned between the
stationary phase survival gene, surE, and a gene encoding a 43 kD
antigenic outer membrane lipoprotein that is highly homologous to
the recently identified bacterial lipoprotein, LppB/NlpD, which has
been associated with virulence (Padmalayam et al., Infect. Immun.,
68: 4972-4979, 2000). Recently, Zhang and coworkers demonstrated
that nlpD and sure gene expression was induced during stationary
phase of bacterial growth in Thermotoga maritima (Zhang et al.,
Structure (Camb), 9: 1095-1106, 2001). Therefore, under
stress-induced conditions in the middle ear, this NTHi lipoprotein
may be expressed. TABLE-US-00004 TABLE 3A Genbank Contig E score
Hit Identity Organism Protein SEQ ID NO: 104 4.00E-59 CpdB
Pasteurella NP_246953.1 nt. 204-659 of multocida SEQ ID NO: 104 106
9.00E-10 hypothetical protein Pyrococcus G71244 nt. 40-309 of
PH0217- horikoshii SEQ ID NO: 106 106 1.00E-08 unknown Pasteurella
NP_246871.1 nt. 605-694 of multocida SEQ ID NO: 106 106 200E-20
Orf122 Chlorobium AAG12204.1 nt. 7-210 of tepidum SEQ ID NO: 106
110 3.00E-05 ArdC antirestriction IncW plasmid pSa AAD52160.1
compliment of protein nt. 959-1162 of SEQ ID NO: 110 110 1.00E-33
hypothetical protein Salmonella NP_458676.1 compliment of enterica
subsp. nt. nt. 181-825 enterica serovar of SEQ ID NO: Typhi 110 111
5.00E-12 putative membrane Salmonella NP_458664.1 compliment of
protein enterica subsp. nt. 45-287 of enterica serovar SEQ ID NO:
111 Typhi 111 6.00E-41 hypothetical protein Salmonella NP_458658.1
compliment of enterica subsp. nt. 1091-1480 of enterica serovar SEQ
ID NO: 111 Typhi 114 7.00E-80 unknown Pasteurella NP_245828.1
compliment of multocida nt. 118-696 of SEQ ID NO: 114 115 2.00E-09
A111R Paramecium NP_048459.1 nt. 555-869 of bursaria Chlorella SEQ
ID NO: 115 virus 1 118 5.00E-45 DNA methylase Vibrio cholerae
NP_231404.1 nt. 44-439 of HsdM, putative SEQ ID NO: 118 122
2.00E-18 unknown Pasteurella NP_245314.1 nt. 865-1302 of multocida
SEQ ID NO: 122 123 4.00E-99 RNA Proteus mirabilis P50509 nt.
351-782 of POLYMERASE SEQ ID NO: 123 SIGMA-32 FACTOR 124 9.00E-58
ACETOLACTATE Spirulina platensis P27868 nt. 603-1025 of SYNTHASE
SEQ ID NO: 124 (ACETOHYDROXY- ACID SYNTHASE) (ALS) 130 0
restriction Neisseria CAA09003.1 nt. 495-1559 of modification
meningitidis SEQ ID NO: 130 system-R protein 131 6.00E-91 uronate
isomerase Salmonella NP_457532.1 compliment of (glucuronate
enterica subsp. nt. 661-1380 of isomerase) enterica serovar SEQ ID
NO: 131 Typhi 133 3.00E-30 GyrA Pasteurella NP_245778.1 compliment
of multocida nt. 1447-1626 of SEQ ID NO: 133 133 1.00E-27 DNA
GYRASE Pectobacterium P41513 compliment of SUBUNIT A carotovorum
nt. 1302-1442 of SEQ ID NO: 133 138 7.00E-06 KicA Pasteurella
NP_245545.1 compliment of multocida nt. 92-157 of SEQ ID NO: 138
138 1.00E-148 TYPE II Haemophilus O30869 compliment of RESTRICTION
aegyptius nt. 164-1045 of ENZYME HAEII SEQ ID NO: 138 (ENDONUCLEASE
HAEII) (R. HAEII) 143 4.00E-06 Gifsy-1 prophage Salmonella
NP_461555.1 compliment of protein typhimurium LT2 nt. 228-632 of
SEQ ID NO: 143 143 1.00E-14 hypothetical protein Bacteriophage
NP_050531.1 compliment of VT2-Sa nt. 778-1248 of SEQ ID NO: 143 143
5.00E-09 hypothetical protein Salmonella CAD09979.1 compliment of
enterica subsp. nt. 715-1026 of enterica serovar SEQ ID NO: 143
Typhi 143 6.00E-10 hypothetical 14.9 kd Escherichia coli
NP_065324.1 nt. 3-173 of protein SEQ ID NO: 143 147 1.00E-38
GTP-binding Escherichia coli NP_289127.1 compliment of elongation
factor, O157:H7 EDL933 nt. 172-342 of may be inner SEQ ID NO: 147
membrane protein 147 2.00E-14 GTP-binding Borrelia NP_212222.1
compliment of membrane protein burgdorferi nt. 17-181 of (IepA) SEQ
ID NO: 147 148 6.00E-17 galactokinase Homo sapiens AAC35849.1
compliment of nt. 746-1246 of SEQ ID NO: 148 148 7.00E-96
GALACTOKINASE Actinobacillus P94169 compliment of (GALACTOSE
pleuropneumoniae nt. 232-741 of KINASE) SEQ ID NO: 148 149 1.00E-92
GTP-binding Buchnera sp. NP_240245.1 compliment of protein
TypA/BipA APS nt. 265-1077 of SEQ ID NO: 149 15 2.00E-21 ORF 1
Escherichia coli CAA39631.1 nt. 665-850 of SEQ ID NO: 15 150
6.00E-17 unknown Pasteurella NP_245919.1 nt. 171-665 of multocida
SEQ ID NO: 150 153 7.00E-07 outer membrane Rickettsia conorii
T30852 nt. 51-623 of protein A SEQ ID NO: 153 155 7.00E-40
cytochrome d Vibrio cholerae NP_233259.1 nt. 583-1002 of ubiquinol
oxidase, SEQ ID NO: 155 subunit II 157 7.00E-13 unknown Pasteurella
NP_245490.1 compliment of multocida nt. 1170-1367 of SEQ ID NO: 157
157 2.00E-05 glycosyl Neisseria AAA68012.1 nt. 85-189 of
transferase gonorrhoeae SEQ ID NO: 157 158 1.00E-152 MltC
Pasteurella NP_246259.1 compliment of multocida nt. 36-530 of SEQ
ID NO: 158 161 3.00E-25 lipoprotein, putative Vibrio cholerae
NP_230232.1 nt. 870-1439 of SEQ ID NO: 161 163 9.00E-53 chorismate
Caulobacter NP_421948.1 nt. 1283-2029 of synthase crescentus SEQ ID
NO: 163 168 3.00E-13 COPPER- Mus musculus Q64430 nt. 66-995 of
TRANSPORTING SEQ ID NO: 168 ATPASE 1 (COPPER PUMP 1) 168 2.00E-22
Cu transporting Homo sapiens 2001422A nt. 135-989 of ATPase P SEQ
ID NO: 168 174 8.00E-48 magnesium/cobalt Mesorhizobium NP_103977.1
nt. 918-1205 of transport protein loti SEQ ID NO: 174 175 5.00E-26
vacB protein Buchnera sp. NP_240369.1 compliment of APS nt. 1-1587
of SEQ ID NO: 175 176 3.00E-21 putative ABC Campylobacter
NP_282774.1 compliment of transport system jejuni nt. 259-1089 of
permease protein [ SEQ ID NO: 176 183 5.00E-29 PROBABLE ATP
Ralstonia NP_521442.1 compliment of SYNTHASE A solanacearum nt.
42-677 of CHAIN SEQ ID NO: 183 TRANSMEMBRANE PROTEIN 185 6.00E-85
putative exported Salmonella NP_458655.1 compliment of protein
enterica subsp. nt. 162-1529 of enterica serovar SEQ ID NO: 185
Typhi 187 2.00E-05 transketolase Homo sapiens AAA61222.1 nt.
709-819 of SEQ ID NO: 187 188 1.00E-116 ribonuclease E Xylella
fastidiosa NP_299884.1 compliment of 9a5c nt. 280-1704 of SEQ ID
NO: 188 192 1.00E-38 ImpA Pasteurella NP_245829.1 nt. 35-448 of
multocida SEQ ID NO: 192 193 3.00E-08 Orf80 Enterobacteria
NP_052285.1 nt. 1612-1818 of phage 186 SEQ ID NO: 193 193 1.00E-06
holin Haemophilus AAC45168.1 nt. 370-576 of somnus SEQ ID NO: 193
193 0.007 unknown Enterobacteria NP_052260.1 nt. 1376-1609 of phage
186 SEQ ID NO: 193 193 2.00E-48 lysozyme Haemophilus AAC45169.1 nt.
608-1093 of somnus SEQ ID NO: 193 199 4.00E-21 unknown protein
Escherichia coli NP_288675.1 nt. 398-778 of O157:H7 SEQ ID NO: 199
EDL933, prophage CP- 933V 199 2.00E-49 hypothetical protein
Bacteriophage NP_049495.1 compliment of 933W nt. 1907-2392 of SEQ
ID NO: 199 20 1.00E-62 RpL14 Pasteurella NP_246344.1 compliment of
multocida nt. 233-601 of SEQ ID NO: 20 200 2.00E-62 hypothetical
protein Salmonella NP_458658.1 compliment of enterica subsp. nt.
431-997 of enterica serovar SEQ ID NO: 200 Typhi 200 3.00E-16
hypothetical protein Salmonella NP_458657.1 compliment of enterica
subsp. nt. 1028-1264 of enterica serovar SEQ ID NO: 200 Typhi 201
2.00E-26 TsaA Pasteurella NP_245732.1 compliment of multocida nt.
1618-1809 of SEQ ID NO: 201 209 6.00E-16 TsaA Pasteurella
NP_245732.1 compliment of multocida nt. 2-136 of SEQ ID NO: 209 211
2.00E-15 unknown Pasteurella NP_245535.1 compliment of multocida
nt. 23-211 of SEQ ID NO: 211 211 1.00E-70 PUTATIVE Ralstonia
NP_520082.1 compliment of ATPASE PROTEIN solanacearum nt. 475-915
of SEQ ID NO: 211 212 3.00E-18 hypothetical protein Escherichia
coli NP_309775.1 compliment of O157:H7 nt. 895-1035 of SEQ ID NO:
212 216 1.00E-173 unknown Pasteurella NP_245069.1 nt. 35-1543 of
multocida SEQ ID NO: 216 217 9.00E-18 diacylglycerol Vibrio
cholerae NP_233101.1 nt. 2083-2208 of kinase SEQ ID NO: 217 221
4.00E-34 Tail-Specific Chlamydia NP_219953.1 nt. 849-1421 of
Protease trachomatis SEQ ID NO: 221 222 4.00E-23 AGR_C_3689p
Agrobacterium NP_355005.1 compliment of tumefaciens str. nt.
940-1305 of C58 (Cereon) SEQ ID NO: 222 224 9.00E-19 unknown
Pasteurella NP_245536.1 nt. 15-308 of multocida SEQ ID NO: 224 225
1.00E-89 portal vector-like Salmonella NP_461651.1 nt. 31-750 of of
protein, in phage typhimurium SEQ ID NO: 225 P2 [Salmonella
LT2Fels-2 typhimurium LT2] prophage 229 2.00E-25 anaerobic
Salmonella CAB62266.1 nt. 1806-2108 of ribonucleotide typhimurium
SEQ ID NO: 229 reductase 234 3.00E-08 conserved Xylella fastidiosa
NP_299850.1 nt. 1680-2048 of hypothetical protein 9a5c SEQ ID NO:
234 234 1.00E-42 Methionine Clostridium NP_348177.1 compliment of
sulfoxide reductase acetobutylicum nt. 415-654 of C-terminal domain
SEQ ID NO: 234 related protein, YPPQ ortholog 235 4.00E-16
phage-related tail Wolbachia AAK85310.1 compliment of protein
endosymbiont of nt. 931-1929 of Drosophila SEQ ID NO: 235
melanogaster 235 6.00E-56 similar to orfG Salmonella NP_461625.1
compliment of protein in phage typhimurium LT2, nt. 313-1863 of
186, Salmonella Fels-2 prophage SEQ ID NO: 235 typhimurium LT2 236
6.00E-20 conserved Pseudomonas NP_252693.1 nt. 1572-1916
hypothetical protein aeruginosa of SEQ ID NO: 236 240 5.00E-27
MODIFICATION Brevibacterium P10283 compliment of METHYLASE BEPI
epidermidis nt. 922-1305 of
SEQ ID NO: 240 241 2.00E-15 phage-related Xylella fastidiosa
NP_299573.1 compliment of protein 9a5c nt. 865-1305 of SEQ ID NO:
241 241 4.00E-08 hypothetical protein phage SPP1 T42296 nt. 73-636
of SEQ ID NO: 241 241 4.00E-07 hypothetical protein Salmonella
NP_458686.1 nt. 10-468 of enterica subsp. SEQ ID NO: 241 enterica
serovar Typhi 242 2.00E-29 translation chloroplast- S35701
compliment of elongation factor soybean nt. 18-1085 of EF-G SEQ ID
NO: 242 247 3.00E-23 GTP Synechococcus Q54769 compliment of
CYCLOHYDROLASE sp. PCC 7942 nt. 1009-1257c I (GTP-CH-I) of SEQ ID
NO: 247 248 6.00E-05 phospho-N- Aquifex aeolicus NP_213025.1 nt.
830-1747 of acetylmuramoyl- SEQ ID NO: 248 pentapeptide-
transferase 25 2.00E-86 PROBABLE Ralstonia NP_522358.1 compliment
of TRANSPORT solanacearum nt. 309-854 of TRANSMEMBRANE SEQ ID NO:
25 PROTEIN 25 7.00E-06 major facilitator Caulobacter NP_419155.1
compliment of family transporter crescentus nt. 134-283 of SEQ ID
NO: 25 250 1.00E-150 CpdB Pasteurella NP_246953.1 compliment of
multocida nt. 36-1016 of SEQ ID NO: 250 252 3.00E-57 alanyl-tRNA
Vibrio cholerae AAA99922.1 compliment of synthetase nt. 1418-1951
of SEQ ID NO: 252 253 1.00E-108 similar to Listeria NP_464432.1
compliment of glutathione monocytogenes nt. 411-1358 of Reductase
EGD-e of SEQ ID NO: 253 259 3.00E-39 hypothetical protein
Salmonella NP_458654.1 compliment of enterica subsp. nt. 342-1037
of enterica serovar SEQ ID NO: 259 Typhi 259 3.00E-17 possible
exported Salmonella NP_458653.1 compliment of protein enterica
subsp. nt. 1251-1607 enterica serovar of SEQ ID NO: Typhi 259 261
5.00E-74 hypothetical protein Haemophilus S27582 compliment of
6-Haemophilus influenzae nt. 3-422 of influenzae SEQ ID NO: 261 263
1.00E-94 putative Haemophilus AAD01406.1 nt. 2142-2672 of
transposase paragallinarum SEQ ID NO: 263 264 1.00E-126 unknown
Actinobacillus NP_067554.1 nt. 40-714 of actinomycetemco SEQ ID NO:
264 mitans 264 1.00E-103 unknown Actinobacillus NP_067555.1 nt.
695-1309 of actinomycetemco SEQ ID NO: 264 mitans 264 2.00E-21
unknown Actinobacillus NP_067556.1 nt. 1302-1448 of actinomycetemco
SEQ ID NO: 264 mitans 265 6.00E-27 Aminopeptidase 2 chloroplast
Q42876 nt. 556-1539 of SEQ ID NO: 265 268 1.00E-116 MutY
Pasteurella NP_246257.1 nt. 1003-1581 of multocida SEQ ID NO: 268
272 1.00E-07 hypothetical protein Bacteriophage NP_049495.1
compliment of 933W nt. 77-232 of SEQ ID NO: 272 274 3.00E-13
unknown Pasteurella NP_246952.1 compliment of multocida nt.
1658-1975 of SEQ ID NO: 274 275 3.00E-20 CafA Neisseria AAG24267.1
nt. 1299-1571 of gonorrhoeae SEQ ID NO: 275 276 1.00E-45 mukE
protein Vibrio cholerae NP_231351.1 compliment of nt. 650-1390 of
SEQ ID NO: 276 276 1.00E-69 KicA Pasteurella NP_245545.1 compliment
of multocida nt. 647-1321 of SEQ ID NO: 276 278 2.00E-56
3-oxoacyl-[acyl- Salmonella NP_455686.1 nt. 1366-1944 of
carrier-protein] enterica subsp. SEQ ID NO: 278 synthase III
enterica serovar Typhi 281 5.00E-56 unknown Pasteurella NP_246261.1
compliment of multocida nt. 31-678 of SEQ ID NO: 281 282 3.00E-09
orf25; similar to T bacteriophage phi NP_490625.1 compliment of
gene of P2 CTX nt. 511-1032 of SEQ ID NO: 282 282 1.00E-08 orf11;
similar to Haemophilus AAC45165.1 compliment of phage P2 gene S-
somnus nt. 1450-1584 of like product, which SEQ ID NO: 282 is
involved in tail synthesis, 282 9.00E-27 putative Salmonella
NP_457167.1 compliment of bacteriophage tail enterica subsp. nt.
3-509 of protein enterica serovar SEQ ID NO: 282 Typhi 286 5.00E-18
plasmid-related Listeria innocua NP_471066.1 compliment of protein
plasmid nt. 887-1501 of SEQ ID NO: 286 287 8.00E-20 GTP Escherichia
coli NP_287920.1 nt. 2-145 of cyclohydrolase II O157:H7 EDL933 SEQ
ID NO: 287 289 1.00E-168 MODIFICATION Haemophilus O30868 compliment
of METHYLASE aegyptius nt. 138-1091 of HAEII SEQ ID NO: 289 289
5.00E-11 TYPE II Haemophilus O30869 compliment of RESTRICTION
aegyptius nt. 22-132 of ENZYME HAEII SEQ ID NO: 289 289 6.00E-47
mukF homolog Haemophilus AAB70828.1 compliment of influenzae
biotype nt. 1107-1385 aegyptius of SEQ ID NO: 289 294 1.00E-171
LICA PROTEIN Haemophilus P14181 compliment of influenzae nt.
677-1564 of RM7004 SEQ ID NO: 294 297 1.00E-158 DNA methylase
Vibrio cholerae NP_231404.1 compliment of HsdM, putative nt.
12-1136 of SEQ ID NO: 297 302 0 HEME-BINDING Haemophilus P33950 nt.
3-1316 of PROTEIN A influenzae DL42 SEQ ID NO: 302 304 6.00E-19
hypothetical protein 6 Haemophilus S27582 nt. 121-267 of influenzae
SEQ ID NO: 304 305 6.00E-40 putative Streptococcus NP_269557.1 nt.
65-805 of recombinase- pyogenes M1 SEQ ID NO: 305 phage associated
GAS 305 7.00E-22 single stranded Shewanella sp. AAB57886.1 nt.
1607-2014 of DNA-binding F1A SEQ ID NO: 305 protein 305 1.00E-43
phage-related Bacillus NP_244410.1 nt. 92-751 of protein halodurans
SEQ ID NO: 305 312 1.00E-28 PUTATIVE Ralstonia NP_518994.1 nt.
1819-2673 of BACTERIOPHAGE- solanacearum SEQ ID NO: 312 RELATED
TRANSMEMBRANE PROTEIN 312 9.00E-25 similar to Homo sapiens
XP_068727.1 nt. 27-1001 of BASEMENT SEQ ID NO: 312 MEMBRANE-
SPECIFIC HEPARAN SULFATE PROTEOGLYCAN CORE PROTEIN PRECURSOR (HSPG)
315 2.00E-45 uracil permease Deinococcus NP_296001.1 compliment of
radiodurans nt. 525-1592 of SEQ ID NO: 315 318 7.00E-15 CzcD
Pasteurella NP_246276.1 compliment of multocida nt. 3-227 of SEQ ID
NO: 318 320 2.00E-60 orf3; similar to Haemophilus AAC45159.1
compliment of endonuclease somnus nt. 606-1241 of subunit of the
SEQ ID NO: 320 phage P2 terminase (gene M) 320 2.00E-09 orf4;
similar to Haemophilus AAC45160.1 compliment of head somnus nt.
52-285 of completion/stabilization SEQ ID NO: 320 protein (gene L)
of phage P2 320 3.00E-35 orf2; similar to Haemophilus AAC45158.1
compliment of major capsid somnus nt. 1271-1624 of protein
precursor of SEQ ID NO: 320 phage P2 (gene N) 323 4.00E-37 dedC
protein Escherichia coli AAA23966.1 compliment of nt. 74-463 of SEQ
ID NO: 323 324 1.00E-153 conserved Neisseria NP_274972.1 compliment
of hypothetical protein meningitidis nt. 930-1943 of MC58 SEQ ID
NO: 324 326 5.00E-52 selenophosphate Eubacterium CAB53511.1
compliment of synthetase acidaminophilum nt. 1186-2292 of SEQ ID
NO: 326 328 1.00E-129 secretion protein Pseudomonas NP_252510.1
compliment of SecD aeruginosa nt. 8-625 of SEQ ID NO: 328 333
3.00E-08 unknown Pasteurella NP_245489.1 compliment of multocida
nt. 5-418 of SEQ ID NO: 333 336 6.00E-38 probable methyl
Pseudomonas NP_253353.1 compliment of transferase aeruginosa nt.
2547-2819 of SEQ ID NO: 336 338 2.00E-98 Pmi Pasteurella
NP_245766.1 nt. 144-842 of multocida SEQ ID NO: 338 339 2.00E-07
tRNA Escherichia coli QQECPE nt. 2331-2540 of
nucleotidyltransferase SEQ ID NO: 339 340 0 DNA gyrase, Salmonella
NP_461214.1 compliment of subunit A, type II typhimurium LT2 nt.
93-1799 of topoisomerase SEQ ID NO: 340 342 4.00E-12 tolA protein
Haemophilus JC5212 nt. 980-1318 of influenzae SEQ ID NO: 342 344
1.00E-07 MODIFICATION Haemophilus P50192 compliment of METHYLASE
parahaemolyticus nt. 849-1034 of HPHIA SEQ ID NO: 344 344 8.00E-05
ABC transporter Leishmania major AAF31030.1 compliment of protein 1
nt. 17-205 of SEQ ID NO: 344 349 3.00E-44 conserved Neisseria
NP_273467.1 compliment of hypothetical protein meningitidis nt.
1397-1903 of MC58 SEQ ID NO: 349 349 8.00E-09 hypothetical protein
Pseudomonas NP_252667.1 compliment of aeruginosa nt. 795-1121 of
SEQ ID NO: 349 349 9.00E-10 conserved Helicobacter NP_207009.1
compliment of hypothetical pylori 26695 nt. 1319-1816 of secreted
protein SEQ ID NO: 349 349 2.00E-06 putative TPR Salmonella
NP_463149.1 compliment of repeat protein typhimurium LT2 nt.
2244-2558 of SEQ ID NO: 349 35 1.00E-23 type I restriction- Xylella
fastidiosa NP_300003.1 compliment of modification 9a5c nt. 29-388
of system specificity SEQ ID NO: 35 determinant 352 1.00E-116
putative peptidase Escherichia coli NP_416827.1 compliment of K12
nt. 951-1640 of SEQ ID NO: 352 352 0 unknown Pasteurella
NP_245275.1 compliment of multocida nt. 86-946 of SEQ ID NO: 352
354 5.00E-86 putative uronate Salmonella NP_462052.1 compliment of
isomerase typhimurium LT2 nt. 168-914 of SEQ ID NO: 354 356
1.00E-07 isomerase-like Escherichia coli S57220 nt. 5-73 of protein
(DsbD)- SEQ ID NO: 356 358 1.00E-07 USG protein Pediococcus
CAC16793.1 nt. 534-1307 of pentosaceus SEQ ID NO: 358 358 0.005
HsdS protein Escherichia coli CAA10700.1 nt. 26-205 of SEQ ID NO:
358 361 1.00E-152 maltodextrin Escherichia coli NP_289957.1
compliment of phosphorylase O157:H7 EDL933 nt. 77-922 of SEQ ID NO:
361 363 6.00E-06 BH2505-unknown Bacillus NP_243371.1 nt. 554-844 of
conserved protein halodurans SEQ ID NO: 363 368 1.00E-12 H02F09.3.p
Caenorhabditis NP_508295.1 compliment of elegans nt. 1069-1977 of
SEQ ID NO: 368 368 6.00E-27 hypothetical Mesorhizobium NP_102360.1
compliment of glycine-rich protein loti nt. 1201-1986 of SEQ ID NO:
368 37 6.00E-09 putative ATP- Escherichia coli NP_415469.1
compliment of binding component K12 nt. 455-691 of of a transport
SEQ ID NO: 37 system 372 7.00E-18 conserved Clostridium BAB80319.1
compliment of hypothetical protein perfringens nt. 1763-1924 of SEQ
ID NO: 372
376 7.00E-24 putative Salmonella NP_456379.1 compliment of
bacteriophage enterica subsp. nt. 158-808 of protein enterica
serovar SEQ ID NO: 376 Typhi 376 8.00E-10 hypothetical protein
Xylella fastidiosa NP_298882.1 compliment of 9a5c nt. 1129-1671 of
SEQ ID NO: 376 376 9.00E-06 lin1713 Listeria innocua NP_471049.1
compliment of nt 913-1557 of SEQ ID NO: 376 377 6.00E-05 Vng1732c
Halobacterium sp. NP_280487.1 nt. 2378-2587 of NRC-1 SEQ ID NO: 377
377 1.00E-11 INVASIN Yersinia P31489 compliment of PRECURSOR
enterocolitica nt. 127-345 of (OUTER SEQ ID NO: 377 MEMBRANE
ADHESIN) 382 4.00E-16 unknown Pasteurella NP_246871.1 compliment of
multocida nt. 967-1068 of SEQ ID NO: 382 383 4.00E-36 putative
Streptomyces BAB69302.1 nt. 488-1162 of transposase avermitilis SEQ
ID NO: 383 383 3.00E-58 recombinase IncN plasmid R46 NP_511241.1
compliment of nt. 1-393 of SEQ ID NO: 383 383 4.00E-24 transposase
Escherichia coli I69674 nt. 1294-1740 of SEQ ID NO: 383 383 0 tnpA
Yersinia CAA73750.1 nt. 1782-2834 of enterocolitica SEQ ID NO: 383
385 2.00E-31 unknown Pasteurella NP_246065.1 nt. 1515-1772 of
multocida SEQ ID NO: 385 386 5.00E-65 cydC [ Escherichia coli
AAA66172.1 compliment of nt. 3438-4115 of SEQ ID NO: 386 386
4.00E-33 ABC transporter, Mesorhizobium NP_105463.1 compliment of
ATP-binding loti nt. 2569-3390 of protein SEQ ID NO: 386 388
3.00E-45 60 KDA INNER- Coxiella burnetii P45650 compliment of
MEMBRANE nt. 3211-3759 PROTEIN of SEQ ID NO: HOMOLOG 388 390
4.00E-25 putative DNA- Salmonella NP_458175.1 nt. 1051-1416 of
binding protein enterica subsp. SEQ ID NO: 390 enterica serovar
Typhi 390 3.00E-13 transcriptional Bacillus NP_241773.1 compliment
of regulator halodurans nt. 84-578 of SEQ ID NO: 390 390 3.00E-06
DNA translocase Staphylococcus NP_372265.1 compliment of stage III
sporulation aureus subsp. nt. 620-871 of prot homolog aureus Mu50
SEQ ID NO: 390 395 7.00E-31 ATPase, Cu++ Homo sapiens NP_000044.1
compliment of transporting, beta nt. 615-1406 of polypeptide SEQ ID
NO: 395 397 3.00E-23 terminase large Bacteriophage NP_112076.1
compliment of subunit HK620 nt. 2363-2725 of SEQ ID NO: 397 397
3.00E-16 hypothetical protein Xylella fastidiosa NP_297824.1
compliment of 9a5c nt. 1517-1744 of SEQ ID NO: 397 398 4.00E-67
orf32 Haemophilus NP_536839.1 compliment of phage HP2 nt. 1288-1866
of SEQ ID NO: 398 398 8.00E-24 putative Salmonella NP_463063.1
compliment of cytoplasmic protein typhimurium LT2 nt. 798-1220 of
SEQ ID NO: 398 398 2.00E-83 orf31 Haemophilus NP_043502.1
compliment of phage HP1 nt. 1881-2510 of SEQ ID NO: 398 399
1.00E-94 HEME/HEMOPEXIN- Haemophilus P45355 nt. 88-774 of BINDING
influenzae N182 SEQ ID NO: 399 PROTEIN 401 3.00E-63 Sty SBLI
Salmonella CAA68058.1 nt. 1690-2742 of enterica SEQ ID NO: 401 401
3.00E-06 RESTRICTION- Mycoplasma NP_325912.1 nt. 79-489 of
MODIFICATION pulmonis SEQ ID NO: 401 ENZYME SUBUNIT M3 402 2.00E-13
OPACITY Neisseria Q05033 compliment of PROTEIN OPA66 gonorrhoeae
nt. 2634-2915 of PRECURSOR SEQ ID NO: 402 406 8.00E-13 type I
restriction Neisseria NP_273876.1 nt. 281-520 of enzyme EcoR124IIR
meningitidis SEQ ID NO: 406 MC58 407 6.00E-65 unknown Pasteurella
NP_246237.1 nt. 938-2450 of multocida SEQ ID NO: 407 407 5.00E-99
PepE Pasteurella NP_245391.1 nt. 1216-1917 of multocida SEQ ID NO:
407 407 1.00E-16 Hemoglobin- Haemophilus Q48153 nt. 1-141 of
haptoglobin binding influenzae Tn106 SEQ ID NO: 407 protein A 409
1.00E-106 hypothetical protein 1 Haemophilus S27577 compliment of
influenzae nt. 2524-3159 of SEQ ID NO: 409 411 4.00E-29
heme-repressible Haemophilus AAB46794.1 nt. 391-615 of
hemoglobin-binding influenzae, type b, SEQ ID NO: 411 protein
strain HI689 411 0 Hemoglobin- Haemophilus Q48153 nt. 651-3263 of
haptoglobin binding influenzae Tn106 SEQ ID NO: 411 protein A 412
2.00E-07 REGULATORY bacteriophage P03036 compliment of PROTEIN CRO
434 nt. 59-259 of (ANTIREPRESSOR) SEQ ID NO: 412 412 4.00E-06
hypothetical protein Bacteriophage CAC83535.1 nt. 1436-1654 of P27
SEQ ID NO: 412 413 8.00E-07 hypothetical protein Deinococcus
NP_294301.1 compliment of radiodurans nt. 791-1012 of SEQ ID NO:
413 414 9.00E-65 conserved Vibrio cholerae NP_230092.1 nt.
1696-2103 of hypothetical protein SEQ ID NO: 414 414 3.00E-93
unknown Pasteurella NP_246834.1 nt. 1777-2109 of multocida SEQ ID
NO: 414 416 2.00E-17 unknown Pasteurella NP_246629.1 compliment of
multocida nt. 2565-2831 of SEQ ID NO: 416 416 4.00E-26 hypothetical
protein Escherichia coli S30728 compliment of o154 nt. 1928-2254 of
SEQ ID NO: 416 416 3.00E-37 transport protein Pseudomonas
NP_253757.1 compliment of TatC aeruginosa nt. 1494-2018 of of SEQ
ID NO: 416 417 1.00E-66 weakly similar to Listeria innocua
NP_471073.1 compliment of methyltransferases nt. 999-1928 of SEQ ID
NO: 417 417 5.00E-05 DNA-BINDING Pectobacterium Q47587 compliment
of PROTEIN RDGA carotovorum nt. 3526-4212 of SEQ ID NO: 417 417
2.00E-29 putative phage- Yersinia pestis NP_407132.1 compliment of
related protein nt. 2546-2938 of SEQ ID NO: 417 417 3.00E-06
Adenine-specific Thermoplasma NP_393798.1 compliment of DNA
methylase acidophilum nt. 826-1020 of SEQ ID NO: 417 43 9.00E-16
PcnB Pasteurella NP_245801.1 nt. 511-870 of multocida SEQ ID NO: 43
434 2.00E-97 beta' subunit of Nephroselmis NP_050840.1 compliment
of RNA polymerase olivacea nt. 32-1534 of SEQ ID NO: 434 435
4.00E-52 MODIFICATION Brevibacterium P10283 compliment of METHYLASE
BEPI epidermidis nt. 11-565 of SEQ ID NO: 435 435 4.00E-57
pentafunctional Saccharomyces NP_010412.1 compliment of arom
polypeptide cerevisiae nt. 757-2064 of (contains: 3- SEQ ID NO: 435
dehydroquinate synthase, 3- dehydroquinate dehydratase (3-
dehydroquinase), shikimate 5- dehydrogenase, shikimate kinase, and
epsp synthase 437 5.00E-70 dihydrofolate Haemophilus S52336 nt.
2393-2767 of reductase influenzae SEQ ID NO: 437 (clinical isolate
R1042) 438 1.00E-106 polyA polymerase Vibrio cholerae NP_230244.1
nt. 3-1124 of SEQ ID NO: 438 439 6.00E-60 Porphyrin Salmonella
NP_457816.1 nt. 2343-2783 of biosynthetic protein enterica subsp.
SEQ ID NO: 439 enterica serovar Typhi 441 5.00E-73 RimM Pasteurella
NP_246234.1 compliment of multocida nt. 151-441 of SEQ ID NO: 441
442 9.00E-31 LIPOPROTEIN Salmonella P40827 compliment of NLPD
typhimurium nt. 3362-3520 of SEQ ID NO: 442 444 6.00E-24 glycine
betaine Staphylococcus NP_371872.1 compliment of transporter aureus
subsp. nt. 2242-2514 of aureus Mu50 SEQ ID NO: 444 452 6.00E-28
unknown Pasteurella NP_245616.1 compliment of multocida nt. 533-883
of SEQ ID NO: 452 452 0 Type I restriction Escherichia coli Q47163
nt. 3291-4154 of enzyme Ecoprrl M SEQ ID NO: 452 protein 452
2.00E-75 type I restriction Ureaplasma NP_077929.1 nt. 4156-4562 of
enzyme M protein urealyticum SEQ ID NO: 452 455 9.00E-56 PROBABLE
Ralstonia NP_520059.1 nt. 2028-2774 of BACTERIOPHAGE solanacearum
SEQ ID NO: 455 PROTEIN 455 2.00E-55 orf2; similar to Haemophilus
AAC45158.1 nt. 2864-3490 of major capsid somnus SEQ ID NO: 455
protein precursor of phage P2 (gene N), 455 1.00E-175 gpP
Enterobacteria NP_046758.1 compliment of phage P2 nt. 127-1812 of
SEQ ID NO: 455 456 1.00E-38 hypothetical protein Pseudomonas
NP_542872.1 compliment of putida nt. 1010-1282 of SEQ ID NO: 456
456 1.00E-172 hypothetical protein Pseudomonas NP_542873.1
compliment of putida nt. 1443-2006 of SEQ ID NO: 546 457 1.00E-116
hypothetical protein Haemophilus S15287 compliment of (galE 5'
region)- influenzae nt. 62-961 of Haemophilus SEQ ID NO: 457
influenzae 457 1.00E-134 dTDPglucose 4,6- Actinobacillus T00102 nt.
2637-3656 of dehydratase actinomycetemco SEQ ID NO: 457 mitans 459
2.00E-10 RNA polymerase Synechocystis sp. NP_441586.1 nt. 25-117 of
gamma-subunit PCC 6803 SEQ ID NO: 459 461 9.00E-51 conserved
Staphylococcus NP_370593.1 nt. 4124-4624 of hypothetical protein
aureus subsp. SEQ ID NO: 461 aureus Mu50 462 9.00E-06 NADH
Burkholderia AAG01016.1 nt. 703-828 of dehydrogenase pseudomallei
SEQ ID NO: 462 465 3.00E-41 GTP-binding Synechocystis sp.
NP_441951.1 compliment of protein Era PCC 6803 nt. 2470-2787 of SEQ
ID NO: 465 466 1.00E-15 putative Salmonella NP_455548.1 nt.
837-1478 of bacteriophage enterica subsp. SEQ ID NO: 466 protein
enterica serovar Typhi 466 1.00E-90 orf31 Haemophilus NP_043502.1
nt. 2396-3199 of phage HP1 SEQ ID NO: 466 469 0 Hemoglobin and
Haemophilus Q9X442 compliment of hemoglobin- influenzae HI689 nt.
427-3459 of haptoglobin binding SEQ ID NO: 469 protein C precursor
471 8.00E-05 transposase, Neisseria NP_274608.1 nt. 2957-3217 of
putative meningitidis SEQ ID NO: 471 MC58 472 6.00E-08 hypothetical
protein Salmonella NP_458660.1 compliment of enterica subsp. nt.
2881-3270 of enterica serovar SEQ ID NO: 472 Typhi 472 5.00E-23
antirestriction Mesorhizobium NP_106707.1 nt. 4908-5324 of protein
loti SEQ ID NO: 472 472 1.00E-75 hypothetical protein Salmonella
NP_458661.1 compliment of enterica subsp. nt. 1931-2776 of enterica
serovar SEQ ID NO: 472 Typhi 472 9.00E-72 hypothetical protein
Salmonella NP_458662.1 compliment of enterica subsp. nt. 544-1689
of enterica serovar SEQ ID NO: 472 Typhi 475 3.00E-25 unknown
Pasteurella NP_244952.1 nt. 3207-3626 of multocida SEQ ID NO: 475
476 8.00E-73 putative DNA- Salmonella NP_458175.1 compliment of
binding protein enterica subsp. nt. 3339-4310 of enterica serovar
SEQ ID NO: 476
Typhi 476 6.00E-47 anticodon nuclease Neisseria NP_273873.1|
compliment of meningitidis nt. 4397-4885 of MC58 SEQ ID NO: 476 478
3.00E-06 methionin Arabidopsis CAB38313.1 compliment of
synthase-like thaliana nt. 3554-3679 of enzyme SEQ ID NO: 478 478
3.00E-05 unknown Pasteurella NP_245444.1 compliment of multocida
nt. 164-250 of SEQ ID NO: 478 479 1.00E-18 conserved Xylella
fastidiosa NP_298841.1 nt. 2302-2658 of hypothetical protein 9a5c
SEQ ID NO: 479 48 3.00E-19 Dca Neisseria AAF12796.1 compliment of
gonorrhoeae nt. 225-746 of SEQ ID NO: 48 482 1.00E-06 hypothetical
protein Neisseria NP_275122.1 nt. 2055-2189 of meningitidis SEQ ID
NO: 482 MC58 482 9.00E-28 conserved Neisseria NP_274383.1 nt.
1689-1898 of hypothetical protein meningitidis SEQ ID NO: 482 MC58
487 5.00E-75 conserved Neisseria NP_284304.1 nt. 2541-2978 of
hypothetical protein meningitidis SEQ ID NO: 487 Z2491 488 2.00E-64
unknown Pasteurella NP_246617.1 nt. 2983-3540 of multocida SEQ ID
NO: 488 488 8.00E-93 1-deoxy-D-xylulose Zymomonas AAD29659.1 nt.
1344-1880 of 5-phosphate mobilis SEQ ID NO: 488 reductoisomerase
491 5.00E-51 rubredoxin Clostridium AAB50346.1 compliment of
oxidoreductase acetobutylicum nt. 1690-2439 of homolog SEQ ID NO:
491 492 1.00E-27 phosphotransferase Staphylococcus AAK83253.1
compliment of system enzyme aureus nt. 755-970 of IIA-like protein
SEQ ID NO: 492 493 2.00E-84 unknown Actinobacillus AAC70895.1 nt.
3333-3935 of actinomycetemco SEQ ID NO: 493 mitans 493 4.00E-49
unknown Helicobacter NP_223898.1 nt. 3345-4010 of pylori J99 SEQ ID
NO: 493 493 9.00E-31 transcriptional Acinetobacter AAF20290.1 nt.
1885-2793 of factor MdcH calcoaceticus SEQ ID NO: 493 493 6.00E-30
HimA Pasteurella NP_245565.1 nt. 1129-1260 of multocida SEQ ID NO:
493 494 4.00E-85 putative prophage Yersinia pestis NP_404712.1 nt.
900-2099 of integrase SEQ ID NO: 494 494 4.00E-63 DNA Xylella
fastidiosa NP_299063.1 compliment of methyltransferase 9a5c nt.
5544-6170 of SEQ ID NO: 494 494 6.00E-19 MODIFICATION Lactococcus
lactis P34877 compliment of METHYLASE subsp. cremoris nt. 5019-6113
of SCRFIA SEQ ID NO: 494 497 0 transferrin-binding Haemophilus
S70906 nt. 3251-4999 of protein 1 influenzae (strain SEQ ID NO: 497
PAK 12085) 50 5.00E-07 AcpP Pasteurella NP_246856.1 nt. 2-136 of
multocida SEQ ID NO: 50 501 7.00E-50 conserved Vibrio cholerae
NP_231403.1 compliment of hypothetical protein nt. 3649-4872 of SEQ
ID NO: 501 501 0 type I restriction Vibrio cholerae NP_231400.1
compliment of enzyme HsdR, nt. 1551-3440 of putative SEQ ID NO: 501
501 4.00E-13 ATP-dependent Deinococcus NP_295921.1 compliment of
DNA helicase radiodurans nt. 5317-5844 of RecG-related SEQ ID NO:
501 protein 501 5.00E-11 conserved Ureaplasma NP_077868.1
compliment of hypothetical urealyticum nt. 5098-5769 of SEQ ID NO:
501 504 2.00E-44 OUTER Haemophilus Q48218 compliment of MEMBRANE
influenzae nt. 4681-5019 of PROTEIN P2 AG30010 SEQ ID NO: 504
PRECURSOR (OMP P2) 507 0 SpoT Pasteurella NP_245857.1 compliment of
multocida nt. 3685-5316 of SEQ ID NO: 507 51 6.00E-87 glucosamine--
Vibrio cholerae NP_230141.1 nt. 30-470 of fructose-6- SEQ ID NO: 51
phosphate aminotransferase (isomerizing) 512 2.00E-28 dipeptide
transport Yersinia pestis NP_407439.1 compliment of system permease
nt. 1095-1580 of protein SEQ ID NO: 512 512 3.00E-82 SapC
Pasteurella NP_245850.1 compliment of multocida nt. 730-1095 of SEQ
ID NO: 512 514 9.00E-06 putative integral Campylobacter NP_281236.1
compliment of membrane protein jejuni nt. 577-684 of SEQ ID NO: 514
514 3.00E-11 orf, hypothetical Escherichia coli NP_286004.1
compliment of protein O157:H7 EDL933 nt. 449-568 of SEQ ID NO: 514
518 0 putative inner Neisseria NP_284893.1 nt. 92-1927 of membrane
transacylase meningitidis SEQ ID NO: 518 protein Z2491 519 4.00E-30
hypothetical protein Mesorhizobium NP_108196.1 compliment of loti
nt. 2221-3159 of SEQ ID NO: 519 519 2.00E-12 conserved Listeria
innocua NP_471067.1 compliment of hypothetical protein nt.
3994-5241 of SEQ ID NO: 519 519 6.00E-20 hypothetical protein
Mesorhizobium NP_108198.1 compliment of loti nt. 707-1552 of SEQ ID
NO: 519 519 4.00E-26 putative Salmonella NP_455526.1 compliment of
bacteriophage enterica subsp. nt. 3982-5163 of protein enterica
serovar SEQ ID NO: 519 Typhi 52 3.00E-94 OUTER Haemophilus Q48218
nt. 45-788 of MEMBRANE influenzae SEQ ID NO: 52 PROTEIN P2
PRECURSOR (OMP P2) 520 0 excision nuclease Escherichia coli
NP_418482.1 compliment of subunit A K12 nt. 6309-7745 of SEQ ID NO:
520 521 5.00E-08 zinc/manganese Rickettsia conorii NP_359651.1 nt.
2236-2652 of ABC transporter SEQ ID NO: 521 substrate binding
protein 521 1.00E-140 unknown Pasteurella NP_245865.1| nt. 338-1390
of multocida SEQ ID NO: 521 521 1.00E-86 ORF_f432 Escherichia coli
AAB40463.1 nt. 203-1390 of SEQ ID NO: 521 522 3.00E-22 unknown
Pasteurella NP_246093.1 nt. 670-885 of multocida SEQ ID NO: 522 526
5.00E-33 exodeoxyribonuclease Yersinia pestis NP_404635.1 nt.
5582-6202 of V alpha chain SEQ ID NO: 526 526 1.00E-62
exodeoxyribonuclease Vibrio cholerae NP_231950.1 nt. 5675-6193 of
V, 67 kDa SEQ ID NO: 526 subunit 527 1.00E-147 unknown Pasteurella
NP_245980.1 nt. 4283-5203 of multocida SEQ ID NO: 527 527 0 Mfd
Pasteurella NP_245978.1 nt. 7545-8759 of multocida SEQ ID NO: 527
527 0 transcription-repair Salmonella NP_455708.1 nt. 7611-8762 of
coupling factor enterica subsp. SEQ ID NO: 527 (TrcF) enterica
serovar Typhi 527 0 PROBABLE Ralstonia NP_519763.1 nt. 7611-8870 of
TRANSCRIPTION- solanacearum SEQ ID NO: 527 REPAIR COUPLING FACTOR
PROTEIN 528 1.00E-48 undecaprenyl Chlamydia NP_297109.1 nt.
2918-3712 of pyrophosphate muridarum SEQ ID NO: 528 synthetase 528
0 leucyl-tRNA Vibrio cholerae NP_230603.1 compliment of synthetase
nt. 180-2822 of SEQ ID NO: 528 529 1.00E-104 DNA PRIMASE Legionella
P71481 compliment of pneumophila nt. 3316-3960 of SEQ ID NO: 529
534 9.00E-29 putative integrase Salmonella NP_461690.1 nt.
4668-5009 of typhimurium LT2 SEQ ID NO: 534 534 6.00E-18
hypothetical protein Neisseria NP_283002.1 compliment of NMA0153
meningitidis nt. 5933-6337 of Z2491 SEQ ID NO: 534 534 2.00E-23
hypothetical protein Deinococcus NP_294868.1 nt. 6908-7654 of
radiodurans SEQ ID NO: 534 534 1.00E-88 prophage CP4-57 Escherichia
coli NP_417111.1 nt. 5057-5875 of integrase K12 SEQ ID NO: 534 535
1.00E-115 phosphate Buchnera sp. NP_240007.1 nt. 3385-4596 of
acetyltransferase APS SEQ ID NO: 535 536 3.00E-35 cobalt membrane
Actinobacillus AAD49727.1 compliment of transport protein
pleuropneumoniae nt. 3531-4136 of CbiQ SEQ ID NO: 536 536 6.00E-37
unknown Pasteurella NP_245305.1 compliment of multocida nt.
6478-6921 of SEQ ID NO: 536 539 2.00E-26 Orf122 Chlorobium
AAG12204.1 compliment of tepidum nt. 1778-2008 of SEQ ID NO: 539
540 1.00E-77 heat shock protein Neisseria NP_273864.1 compliment of
HtpX meningitidis nt. 2567-3481 of MC58 SEQ ID NO: 540 541 0 IleS
Pasteurella NP_246601.1 nt. 3167-4549 of multocida SEQ ID NO: 541
545 2.00E-09 DNA-BINDING Pectobacterium Q47588 nt. 3816-3977 of
PROTEIN RDGB carotovorum SEQ ID NO: 545 545 2.00E-11 putative
Sinorhizobium NP_437741.1 compliment of transposase meliloti nt.
2786-3019 of SEQ ID NO: 544 545 2.00E-07 Hypothetical 42.5 kd
Escherichia coli BAA77933.1 compliment of protein in thrW- nt.
2614-2811 of argF intergenic SEQ ID NO: 545 region 545 4.00E-18
putative IS element Salmonella NP_454711.1 nt. 1955-2230 of
transposase enterica subsp. SEQ ID NO: 545 enterica serovar Typhi
546 0 HEME/HEMOPEXIN- Haemophilus P45354 nt. 5551-7809 of BINDING
influenzae SEQ ID NO: 546 PROTEIN 546 0 HEME/HEMOPEXIN Haemophilus
P45356 nt. 3842-5536 of UTILIZATION influenzae SEQ ID NO: 546
PROTEIN B 546 0 HEME/HEMOPEXIN Haemophilus P45357 nt. 1638-3176 of
UTILIZATION influenzae SEQ ID NO: 546 PROTEIN C 546 2.00E-12 HasR
Pasteurella NP_246561.1 nt. 3149-3763 of multocida SEQ ID NO: 546
549 0 unknown Pasteurella NP_246821.1 nt. 2526-3512 of multocida
SEQ ID NO: 549 549 1.00E-121 putative membrane Yersinia pestis
NP_404859.1 nt. 605-1108 of protein SEQ ID NO: 549 549 0 unknown
Pasteurella NP_246822.1 nt. 1122-1664 of multocida SEQ ID NO: 549
551 1.00E-157 type I restriction- Xylella fastidiosa NP_300016.1
compliment of modification 9a5c nt. 7396-8322 of system SEQ ID NO:
551 endonuclease 552 1.00E-100 valyl-tRNA Deinococcus NP_293872.1
compliment of synthetase radiodurans nt. 6691-8688 of SEQ ID NO:
552 552 0 VALYL-TRNA Haemophilus P36432 compliment of SYNTHETASE
parainfluenzae nt. 5850-6647 of SEQ ID NO: 552 553 0 DNA-directed
RNA Vibrio cholerae NP_229982.1 nt. 2668-6699 of polymerase, beta
SEQ ID NO: 553 subunit 554 0 iron utilization Haemophilus T10887
nt. 991-2508 of protein B influenzae SEQ ID NO: 554 559 1.00E-100
PREPROTEIN Bacillus firmus P96313 nt. 3420-4472 of TRANSLOCASE SEQ
ID NO: 559 SECA SUBUNIT 56 2.00E-23 RpL30 Pasteurella NP_246336.1
compliment of multocida nt. 656-832 of SEQ ID NO: 56 56 9.00E-13
RpS5 Pasteurella NP_246337.1 compliment of multocida nt. 843-1064
of SEQ ID NO: 56 560 1.00E-157 Na+/H+ antiporter Vibrio cholerae
NP_231535.1 2 compliment of nt. 279-2989 of SEQ ID NO: 560 562
1.00E-72 putative biotin Yersinia pestis NP_404419.1 nt. 7862-8878
of
sulfoxide reductase 2 SEQ ID NO: 562 562 1.00E-125 restriction
Neisseria CAA09003.1 nt. 2-985 of modification meningitidis SEQ ID
NO: 562 system-R protein 563 0 IMMUNOGLOBULIN Haemophilus P45384
compliment of A1 PROTEASE influenzae HK715 nt. 4127-9508 of SEQ ID
NO: 563 563 0 3- Schizosaccharomyces O14289 nt. 1980-3983 of
ISOPROPYLMALATE pombe SEQ ID NO: 563 DEHYDRATASE (IPMI) 564
2.00E-79 orf32 Haemophilus NP_536839.1 nt. 6241-6831 of phage HP2
SEQ ID NO: 564 564 7.00E-33 probable variable Salmonella
NP_457882.1 nt. 3707-4177 of tail fibre protein enterica subsp. SEQ
ID NO: 564 enterica serovar Typhi 564 2.00E-14 M protein
Enterobacteria NP_052264.1 nt. 1905-2213 of phage 186 SEQ ID NO:
564 564 4.00E-44 similar to tail fiber Salmonella NP_461635.1 nt.
3171-3692 of protein (gpH) in typhimurium LT2, SEQ ID NO: 564 phage
P2 Fels-2 prophage 564 2.00E-85 gpJ Enterobacteria NP_046773.1 nt.
2267-3166 of phage P2 SEQ ID NO: 564 564 1.00E-24 hypothetical
protein Neisseria NP_284534.1 nt. 6852-7334 of meningitidis SEQ ID
NO: 564 Z2491 564 4.00E-26 gpV Enterobacteria NP_046771.1 nt.
1337-1912 of phage P2 SEQ ID NO: 564 564 2.00E-47 similar to
Escherichia coli BAA16182.1 nt. 11383-11961 [SwissProt P44255 of
SEQ ID NO: 564 564 2.00E-51 hypothetical protein Neisseria
NP_284066.1 nt. 10452-11180 NMA1315 meningitidis of SEQ ID NO:
Z2491 564 564 0 orf31 Haemophilus NP_043502.1 nt. 4160-6226 of
phage HP1 SEQ ID NO: 564 564 2.00E-09 rep Haemophilus NP_536816.1
compliment of phage HP2 nt. 9986-10234 of SEQ ID NO: 564 565
2.00E-57 resolvase/integrase- Haemophilus AAL47097.1 nt.
11885-12445 like protein influenzae biotype of SEQ ID NO: aegyptius
565 565 1.00E-93 integrase Actinobacillus AAC70901.1 compliment of
actinomycetemco nt. 4118-4900 mitans of SEQ ID NO: 565 565 6.00E-35
probable phage Salmonella NP_458745.1 compliment of integrase
enterica subsp. nt. 4148-4990 of enterica serovar SEQ ID NO: 565
Typhi 565 1.00E-107 hypothetical protein Xylella fastidiosa
NP_299042.1 compliment of 9a5c nt. 5066-6817 of SEQ ID NO: 565 566
1.00E-126 hypothetical protein Haemophilus S15287 compliment of
(galE 5' region)- influenzae nt. 10726-11607 of SEQ ID NO: 566 567
0 unknown Pasteurella NP_246387.1 nt.5343-7688 of multocida SEQ ID
NO: 567 568 1.00E-151 multidrug Escherichia coli NP_311575.1 nt.
6-1403 of resistance O157:H7 SEQ ID NO: 568 membrane translocase
568 1.00E-141 YhbX/YhjW/YijP/YjdB Neisseria |NP_275002.1 compliment
of family protein meningitidis nt. 11213-12634 MC58 of SEQ ID NO:
568 570 1.00E-180 hypothetical protein Haemophilus S71024
compliment of 3 (ksgA-lic2B influenzae (strain nt. 12845-13720
intergenic region) RM7004) of SEQ ID NO: 570 571 0
glycerophosphodiester Haemophilus A43576 nt. 1656-2693 of
phosphodiesterase influenzae (isolate SEQ ID NO: 571 772) 571
1.00E-137 outer membrane Haemophilus A43604 nt. 6145-6909 of
protein P4 influenzae SEQ ID NO: 571 precursor- Haemophilus
influenzae 571 2.00E-72 CG8298 gene Drosophila AAF58597.1 nt.
3813-5339 of product [alt 1] melanogaster SEQ ID NO: 571 572
1.00E-40 hypothetical protein Chlamydia G81737 nt. 3734-4099 of
TC0130 muridarum (strain SEQ ID NO: 572 Nigg) 572 5.00E-10
hypothetical protein Pyrococcus NP_142215.1 nt. 4472-4888 of
horikoshii SEQ ID NO: 572 572 3.00E-11 109aa long Sulfolobus
NP_377117.1 nt. 7303-7470 of hypothetical protein tokodaii SEQ ID
NO: 572 572 8.00E-43 hypothetical protein Chlamydophila NP_445524.1
nt. 4289-4618 of pneumoniae SEQ ID NO: 572 AR39 572 9.00E-08 CDH1-D
Gallus gallus AAL31950.1 nt. 7183-7521 of SEQ ID NO: 572 575
1.00E-173 topoisomerase B Salmonella NP_458624.1 nt. 18980-20923
enterica subsp. of SEQ ID NO: enterica serovar 575 Typhi 575
1.00E-100 DNA helicase Salmonella NP_458617.1 nt. 10399-11706
enterica subsp. of SEQ ID NO: enterica serovar 575 Typhi 65
2.00E-53 Sufl Pasteurella NP_245041.1 nt. 3-821 of multocida SEQ ID
NO: 65 67 4.00E-39 putative MFS Salmonella NP_462786.1 compliment
of family tranport typhimurium LT2 nt. 125-1033 of protein (1st
mdule) SEQ ID NO: 67 7 4.00E-29 putative membrane Salmonella
NP_458664.1 compliment of protein enterica subsp. nt. 2-559 of
enterica serovar SEQ ID NO: 7 Typhi 72 2.00E-51 serine transporter
Vibrio cholerae NP_230946.1 nt. 18-803 of SEQ ID NO: 72 74 3.00E-90
hypothetical 21.8K Haemophilus JH0436 compliment of protein (in
locus influenzae nt. 248-766 of involved in SEQ ID NO: 74
transformation)- 77 2.00E-18 RecX protein Legionella CAC33485.1 nt.
480-920 of pneumophila SEQ ID NO: 77 82 4.00E-95 unknown
Pasteurella NP_246414.1 nt. 128-955 of multocida SEQ ID NO: 82 83
2.00E-66 unknown Pasteurella NP_246777.1 nt. 5-556 of multocida SEQ
ID NO: 83 83 6.00E-33 CTP SYNTHASE Helicobacter NP_223042.1
compliment of pylori J99 nt. 1027-1338 of SEQ ID NO: 83. 83
4.00E-34 CTP synthase Campylobacter NP_281249.1 compliment of
jejuni nt. 1024-1275 of SEQ ID NO: 83 84 1.00E-16 REPRESSOR
Bacteriophage P14819 nt. 823-1233 of PROTEIN CI phi-80 SEQ ID NO:
84 84 2.00E-05 orf, hypothetical Escherichia coli NP_415875.1
compliment of protein K12 nt. 533-700 of SEQ ID NO: 84 84 4.00E-11
orf33 bacteriophage phi NP_490633.1 compliment of CTX nt. 32-466 of
SEQ ID NO: 84 85 3.00E-42 SpoT Pasteurella NP_245857.1 nt. 899-1261
of multocida SEQ ID NO: 85 90 1.00E-103 putative methylase
Bacteriophage NP_108695.1 compliment of Tuc2009 nt. 478-1206 of SEQ
ID NO: 90 90 4.00E-11 probable adenine Thermoplasma NP_394624.1
compliment of specific DNA acidophilum nt. 397-1140 of
methyltransferase SEQ ID NO: 90
[0093] TABLE-US-00005 TABLE 3B Full Length Nucleotide Amino Acid
Homology to Genbank Hit Identity Sequence Sequence Location in
Contig Protein CpdB SEQ ID NO: SEQ ID NO: nt. 38041-36068 of
NP_246953.1 686 687 SEQ ID NO: 681 (contig 14) putative membrane
SEQ ID NO: SEQ ID NO: nt. 906601-908094 NP_458664.1 protein 688 689
of SEQ ID NO: 685 (contig 18) GTP-binding SEQ ID NO: SEQ ID NO: nt.
42557-40995 of NP_240245.1 protein TypA/BipA 690 691 SEQ ID NO: 683
(contig 16) outer membrane SEQ ID NO: SEQ ID NO: nt. 7000420-704187
T30852 protein A 692 693 of SEQ ID NO: 685 (contig 18) vacB protein
SEQ ID NO: SEQ ID NO: nt. 39184-36836 of NP_240369.1 694 695 SEQ ID
NO: 683 (contig 16) putative ABC SEQ ID NO: SEQ ID NO: nt.
59155-58370 of NP_282774.1 transport system 696 697 SEQ ID NO: 685
permease protein [ (contig 18) putative exported SEQ ID NO: SEQ ID
NO: nt. 901142-902542 NP_458655.1 protein 698 699 of SEQ ID NO: 685
(contig 18) ImpA SEQ ID NO: SEQ ID NO: nt. 348187-347747
NP_245829.1 700 701 of SEQ ID NO: 685 (contig 18) TsaA SEQ ID NO:
SEQ ID NO: nt. 74941-75548 of NP_245732.1 702 703 SEQ ID NO: 684
(contig 17) PROBABLE SEQ ID NO: SEQ ID NO: nt. 74436-75176 of
NP_522358.1 TRANSPORT 704 705 SEQ ID NO: 685 TRANSMEMBRANE (contig
18) PROTEIN SEQ ID NO: SEQ ID NO. nt. 75160-75660 of 706 707 SEQ ID
NO: 685 (contig 18) possible exported SEQ ID NO: SEQ ID NO: nt.
899618-900262 NP_458653.1 protein 708 709 of SEQ ID NO: 685 (contig
18) LICA PROTEIN SEQ ID NO: SEQ ID NO: nt. 356917-355958 P14181 710
711 of SEQ ID NO: 685 (contig 18) HEME-BINDING SEQ ID NO: SEQ ID
NO: NT. 26114-27739 P33950 PROTEIN A 712 713 of SEQ ID NO: 683
(contig 16) similar to SEQ ID NO: SEQ ID NO: nt. 311610-312683
XP_068727.1 BASEMENT 714 715 of SEQ ID NO: 685 MEMBRANE- (contig
18) SPECIFIC HEPARAN SULFATE PROTEOGLYCAN CORE PROTEIN PRECURSOR
(HSPG) CzcD SEQ ID NO: SEQ ID NO: nt. 34865-35542 of NP_246276.1
716 717 SEQ ID NO: 681 (contig 14) conserved SEQ ID NO: SEQ ID NO:
nt. 194993-193977 NP_274972.1 hypothetical protein 718 719 of SEQ
ID NO: 685 (contig 18) secretion protein SEQ ID NO: SEQ ID NO: nt.
203707-201857 NP_252510.1 SecD 720 721 of SEQ ID NO: 683 (contig
17) ABC transporter SEQ ID NO: SEQ ID NO: nt. 3943-5859 of
AAF31030.1 protein 1 722 723 SEQ ID NO: 681 (contig 14) conserved
SEQ ID NO: SEQ ID NO: nt. 331090-331749 NP_273467.1 hypothetical
protein 724 725 of SEQ ID NO: 685 (contig 18) SEQ ID NO: SEQ ID NO:
nt. 331938-332492 726 727 of SEQ ID NO: 685 (contig 18) SEQ ID NO:
SEQ ID NO: nt. 332681-33232 728 729 of SEQ ID NO: 685 (contig 18)
INVASIN SEQ ID NO: SEQ ID NO: nt. 416757-417020 P31489 PRECURSOR
730 731 of SEQ ID NO: 685 (OUTER (contig 18) MEMBRANE ADHESIN)
HEME/HEMOPEXIN- SEQ ID NO: SEQ ID NO: nt. 229430-232195 P45355
BINDING 732 733 of SEQ ID NO: 384 PROTEIN (contig 17) OPACITY SEQ
ID NO: SEQ ID NO: nt. 375592-375879 Q05033 PROTEIN OPA66 734 735 of
SEQ ID NO: 384 PRECURSOR (contig 17) Hemoglobin- SEQ ID NO: SEQ ID
NO: nt. 45709-42566 of Q48153 haptoglobin binding 736 737 SEQ ID
NO: 681 protein A (contig 14) transport protein SEQ ID NO: SEQ ID
NO: nt. 134452-135222 NP_253757.1 TatC 738 739 of SEQ ID NO: 384
(contig 17) LIPOPROTEIN SEQ ID NO: SEQ ID NO: nt. 18895-20112 of
P40827 NLPD 740 741 SEQ ID NO: 682 (contig 15) Hemoglobin and SEQ
ID NO: SEQ ID NO: nt. 34181-31041 of Q9X442 hemoglobin- 742 743 SEQ
ID NO: 682 haptoglobin binding (contig 15) protein C precursor HimA
SEQ ID NO: SEQ ID NO: nt. 382795-383085 NP_245565.1 744 745 of SEQ
ID NO: 685 (contig 18) transferrin-binding SEQ ID NO: SEQ ID NO:
nt. 178537-175799 S70906 protein 1 746 747 of SEQ ID NO: 683
(contig 16) SapC SEQ ID NO: SEQ ID NO: nt. 197754-196867
NP_245850.1 748 749 of SEQ ID NO: 685 (contig 18) heat shock
protein SEQ ID NO: SEQ ID NO: nt. 40414-41265 of NP_273864.1 HtpX
750 751 SEQ ID NO: 682 (contig 15) HEME/HEMOPEXIN- SEQ ID NO: SEQ
ID NO: nt. 229430-232195 P45354 BINDING 752 753 of SEQ ID NO: 684
PROTEIN (contig 17) HEME/HEMOPEXIN SEQ ID NO: SEQ ID NO: nt.
227721-229418 P45356 UTILIZATION 754 755 of SEQ ID NO: 684 PROTEIN
B (contig 17) HEME/HEMOPEXIN SEQ ID NO: SEQ ID NO: nt.
225516-227645 P45357 UTILIZATION 756 757 of SEQ ID NO: 684
NP_246561.1 PROTEIN C (contig 17) iron utilization SEQ ID NO: SEQ
ID NO: nt. 32076-33611 of T10887 protein B 758 759 SEQ ID NO: 684
(contig 17) PREPROTEIN SEQ ID NO: SEQ ID NO: nt. 82314-84785 of
P96313 TRANSLOCASE 760 761 SEQ ID NO: 683 SECA SUBUNIT (contig 16)
IMMUNOGLOBULIN SEQ ID NO: SEQ ID NO: nt. 171647-166263 P45384 A1
PROTEASE 762 763 of SEQ ID NO: 683 (contig 16) multidrug SEQ ID NO:
SEQ ID NO: nt. 74524-72992 of NP_311575.1 resistance 764 765 SEQ ID
NO: 683 membrane (contig 16) translocase YhbX/YhjW/YijP/YjdB SEQ ID
NO: SEQ ID NO: nt. 61734-63200 of NP_275002.1 family protein 766
767 SEQ ID NO: 683 (contig 16) putative membrane SEQ ID NO: SEQ ID
NO: nt. 906601-908094 NP_458664.1 protein 768 769 of SEQ ID NO: 685
(contig 18) putative membrane SEQ ID NO: SEQ ID NO: nt. 16185-17942
of NP_404859.1 protein 770 771 SEQ ID NO: 683 (contig)
EXAMPLE 3
Construction of the NTHi Promoter Trap Library
[0094] To identify potential virulence determinants of NTHi,
bacterial gene expression was monitored by differential
fluorescence induction (DFI) during early disease progression in
one specific anatomical niche of a chinchilla model of NTHi-induced
otitis media (OM). Genomic DNA fragments from NTHi strain 86-028NP
were cloned upstream of the promoterless gfpmut3 gene using a
promoter trap library. Plasmid pGZRS39A, a derivative of pGZRS-1
isolated from Actinobacillus pleuropneumoniae, is an A.
pleuropneumoniae-Escherichia coli shuttle vector. This plasmid
contains the origin of replication from A. pleuropneumoniae, the
lacZa gene from pUC19 and the kanamycin resistance gene from Tn903.
(West et al., Genes, 160: 81-86, 1995).
[0095] The promoter trap vector was constructed by cloning the GTP
mutant gfpmut3 gene, as a BamHI to EcoRI fragment into pGZRS-39A to
form pRSM2167. This mutant GTP gene contains two amino acid
changes, S65G and S72A, that enhance fluorescence emission when
excited at 488 nm. This mutant also has high solubility and fast
kinetics of chromophore formation (Cormack et al., Gene, 173:
33-38, 1996). This plasmid was transformed by electroporation into
NTHi strain 86-028NP, generating the parent-plasmid strain
86-028NP/pRSM2169.
[0096] Random genomic DNA fragments (described in Example 1) were
prepared for ligation into the promoter probe vector. Genomic DNA
was isolated from strain 86-028NP using the Puregene DNA isolation
kit (Gentra Systems, Minneapolis, Minn.) according to the
manufacturer's protocol. Due to restriction barriers, it was
necessary to isolate the plasmid DNA and use this for the library
generation. The isolated DNA was partially digested with Sau3AI
(NEB, Beverly, Mass.; 0.25 units/.mu.g DNA) for 1 hour at
37.degree. C., separated by gel electrophoresis and DNA fragments
0.5-1.5 kb in size were recovered using the Qiagen gel extraction
kit. For vector preparation, pRSM2167 was isolated from an
overnight culture using the Wizard Plus Maxiprep DNA purification
system (Promega, Madison Wis.) according to the manufacturer's
protocol.
[0097] Plasmid DNA was linearized by BamHI digestion and 5'
phosphate groups removed by treatment with calf intestinal alkaline
phosphatase (CIAP; GibcoBRL Life Technologies). Genomic DNA
fragments were ligated with the linearized, phosphatase-treated
vector and electroporated into competent NTHi strain 86-028NP
prepared for electroporation according to a modified protocol
(Mitchell et al., Nucleic Acids Res., 19: 3625-3628, 1991). When
plasmid DNA was electroporated back into NTHi strain 86-028NP,
transformation efficiency was improved by one-thousand fold.
Briefly, cells were grown to an OD.sub.600=0.3 in sBHI (brain heart
infusion) broth at 37.degree. C., 220 rpm. Cells were chilled on
ice for 30 minutes and subsequently washed with an equal volume of
0.5.times.SG (1.times.SG: 15% glycerol, 272 mM sucrose) at
4.degree. C. Washes were repeated a total of three times.
Subsequently, the cells were diluted in 1.times.SG to a 100.times.
concentrated volume. The cells were electroporated using the BioRad
Gene Pulser II set at 200 ohms, 2.5 kV and 25 .mu.F and then
diluted in 1 ml prewarmed sBHI, incubated for 2 hours at 37.degree.
C., 5% CO.sub.2 and plated on chocolate agar for overnight growth
of transformants.
[0098] Transformants were selected and frozen in pools of 1000
clones in skim milk containing 20% glycerol (vol/vol). A 68,000
member gfp promoter probe library was generated. Using the
probability calculation of Clarke and Carbon (Cell, 9: 91-99,
1976), to achieve a 99% probability of having a given DNA sequence
represented in a library of 300 bp fragments of strain 86-028NP DNA
(1.8.times.10.sup.6 bp/genome), a library of 27,629 clones was
needed. Therefore the present library represents 2.5 fold coverage
of the 86-028NP genome.
[0099] In order to assess the quality of the library, fifty clones
were selected at random, grown overnight on chocolate agar and the
plasmids were isolated and insert DNA sequenced. A majority (64%)
of the selected clones had insert sizes ranging between 200 and 500
bp while 32% exceeded 500 bp. The majority of inserts showed
homology to unique H. influenzae strain Rd open reading frames
(ORFs), and 15 clones had sequence unique to strain 86-028NP DNA.
Of those clones with homology to strain Rd, 60% were in the correct
orientation, 36% of which contained sequence upstream an ORF.
Although a majority of clones had an insert size less than 500 bp,
no correlation was found between small insert size and increased
GFP expression. In fact four clones exhibited slight to moderate
fluorescence in vitro, 3 of which had insert sizes between 200-500
base pairs and one had an insert that was greater than 700 base
pairs.
[0100] A fraction of the library (approximately 1000 clones) was
grown on chocolate agar, harvested in PBS and analyzed by flow
cytometry for GFP fluorescence. Compared to strain
86-028NP/pRSM2169 that contains the promoter trap vector without
insert DNA, the pool of library clones displays an increased
fluorescence intensity. Thus, the library contains clones with
promoters at varying levels of activity.
EXAMPLE 4
Analysis of 86-028NP Derivatives Expressing GFP
[0101] In order to establish the FACS parameters necessary to
identify and sort gfp-expressing bacteria, a panel of isolates
demonstrating varying levels of gfp expression was utilized.
Background fluorescence was assessed using strain 86-028NP/pRSM2169
(negative control), therefore any observed fluorescence would be
due to the lacZ promoter driving gfp expression. However, this
strain does not produce detectable levels of GFP and in fact, does
not demonstrate increased fluorescence when compared to the parent
strain 86-028NP. A high-level gfp-expressing isolate was generated
by cloning a 500 bp fragment containing the strong promoter for
outer membrane protein P2 expression into SalI-BamHI digested
pRSM2167. This plasmid was transformed into 86-028NP by
electroporation, generating the high-level gfp expressing strain
86-028NP/pRSM2211 (highly fluorescent control). This strain
demonstrated an approximate 100 fold increase in GFP fluorescence
compared to strain 86-028NP/pRSM2169. An intermediate fluorescent
derivative clone, 86-028NP/pKMM4B5 (intermediate fluorescent
control), was isolated by FACS analysis and used both in
preliminary experiments and as a control for cell sorting. The DNA
fragment containing a promoter driving gfp expression in vitro is
unique to strain 86-028NP, having no known homology to DNA of other
organisms. This clone exhibits an approximate 10 fold increase in
fluorescence compared to strain 86-028NP/pRSM2169.
[0102] The control strains were resuspended from growth on
chocolate agar and labeled with cross-reactive Phycoprobe R-PE
anti-human IgG (H+L) antibody (10 .mu.g/ml in 100 .mu.l PBS;
Biomeda Corp) for 30 minutes at 4.degree. C. Following three
successive washes to remove unbound antibody, bacteria were
resuspended in 300 .mu.l DPBS for FACS analysis. These control
preparations were used to set the appropriate size and fluorescence
gates using a Coulter Epics Elite flow cytometer (Coulter Corp.)
equipped with an argon laser emitting at 488 nm. Bacteria were
gated for size based on log forward angle and side scatter
detection and for sorting by FITC/PE labeling of bacteria. Sorted
cells were collected into cold sBHI and plated on chocolate agar.
After overnight growth, cells were collected for a secondary round
of infection or were individually selected and grown overnight,
screened by individual clone for fluorescence when grown in vitro,
and frozen in skim milk containing 20% (vol/vol) glycerol prior to
plasmid isolation and sequencing of insert DNA. Sorting efficiency
of control strains was confirmed using a Coulter EPICS flow
cytometer (Coulter Corp.).
[0103] Many plasmids were segregated rapidly in vitro in the
absence of antibiotic selection. Thus, in order to assess whether
the promoter trap vector used here was prone to this event, a
single colony of strain 86-028NP/pRSM2211 (highly fluorescent
control) was isolated on chocolate agar and passaged 20 times in
the absence of antibiotic selection. No significant decrease in
fluorescence intensity was observed when compared to bacteria grown
in the presence of antibiotic. In addition, the plasmid is
maintained in the absence of antibiotic selection in vivo. Similar
bacterial counts were observed when bacteria-containing middle ear
fluids collected from a chinchilla were plated on chocolate agar
with or without kanamycin. These data demonstrate that the promoter
trap vector was stably maintained in the absence of antibiotic
selection.
[0104] In addition to problems with plasmid stability, early
studies on the use of GFP as a reporter to study host-pathogen
interactions demonstrated that GFP could be continuously
synthesized as a cytoplasmic protein with low toxicity, having
minimal effects on the bacterial cell-surface dynamics (Chalfie et
al., Science, 263: 802-805, 1994). The construction of a high level
gfp-expressing derivative allowed the assessment of the GFP
toxicity on NTHi. Growth curves of both the wild-type strain
(86-028NP) and the high GFP producing strain 86-028NP/pRSM2211 were
compared when grown under similar conditions. The growth rates were
similar, indicating that GFP expression was not toxic to the
cells.
[0105] The 86-028NP gfp-expressing derivatives were used to define
the parameters for efficient cell sorting. Strain 86-028NP/pRSM2169
was mixed with the intermediate gfp-expressing derivative, strain
86-028NP/pKMM4B5, at a 100:1 ratio, simulating the in vivo
environment that is expected to contain a small percentage of
gfp-expressing clones relative to the total bacterial population.
This mixture was subjected to FACS analysis, collecting the 1.8%
most fluorescent population and the 52% least fluorescent
population. Flow cytometric analysis of the sorted populations
revealed an enrichment of strain 86-028NP/pKMM4B5 to 65% of the
bacterial population, a phenomenon that was not observed when
sorting on the negative population. Subsequent rounds of sorting
would be expected to further enrich for this intermediate
fluorescent population. The inability to decrease the amount of
fluorescent bacteria in the negative sort was attributed to the
size of the gate set for negative sorting. GFP-negative cells were
enriched by gating on the 10% least fluorescent population.
EXAMPLE 5
Direct Labeling of Bacteria from Middle Ear Fluids
[0106] A similar strategy (as described in Example 5) was applied
to sort fluorescent clones from effusions obtained from the
chinchilla middle ear during AOM. Our ability to use differential
fluorescence induction (DFI) in vivo was dependent upon our ability
to sort gfp-expressing bacteria from non-fluorescent bacteria,
fluorescent and non-fluorescent cellular debris, and eukaryotic
cells.
[0107] Healthy adult chinchillas (Chinchilla lanigera) with no
evidence of middle ear infection by either otoscopy or tympanometry
were used to screen the library for promoter activity in vivo. Two
pools of the NTHi/pRSM2169 library (1000 clones each) were grown
overnight on chocolate agar containing kanamycin. The library was
combined and diluted in cold 10 mM sterile PBS to
3.3.times.10.sup.6 CFU/ml and 300 .mu.l (1.0.times.10.sup.6 CFU;
500 CFU/clone) was used to inoculate the left and the right
chinchilla transbullar cavity (2000 clones/ear). OM development was
monitored by video otoscopy and tympanometry at 24 and 48 hours.
The bacteria multiplied in the middle ear cavity, reaching a
concentration 500 times the inoculum dose by 48 hours as expected
(Bakaletz et al., Infect. Immunity 7: 2746-62, 1999). This
bacterial adaptation to the host environment results in an
inflammatory response, indicated by erythema, vessel dilation and
bulging of the tympanic membrane, infiltration of polymorphonuclear
cells (PMN's), and accumulation of fluid in the middle ear cavity
as observed by otoscopy and microscopic examination of recovered
effusions. Twenty-four and 48 hours later, middle ear fluids were
retrieved by epitympanic tap, and prepared for FACS.
[0108] It is important to note that this analysis was limited to
those bacteria recoverable in the middle ear fluid. In some cases
it was necessary to lavage the middle ear cavity to collect the
bacteria for FACS analysis. Thus, this analysis includes genes
up-regulated when NTHi are loosely adherent to mucosae. NTHi has
been observed to form a biofilm in the middle ear cavity in a
chinchilla model of OM (Erhlich et al., JAMA, 287: 1710-5, 2002).
Since the protocols described herein select for clones recovered
from the planktonic population, it is not expected to recover those
clones in which genes are up-regulated when the bacteria are
associated with mucosal biofilms. Homogenization of middle ear
mucosae and subsequent bacterial cell isolation however, would
enable us to recover these clones. It is also possible that some
GFP-expressing clones were recovered in the effusion, yet were
adherent to eukaryotic cells present in the effusion as exfoliated
cells, or in aggregates. These bacteria are difficult to recover
from the effusion without compromising the sorting efficiency.
Therefore the middle ear fluids were treated with a mucolytic
agent, then centrifuged to remove large aggregates and eukaryotic
cells and prior to labeling.
[0109] Chinchilla middle ear fluids were diluted, if necessary, to
250 .mu.l with sterile saline. An equal volume of
N-acetyl-L-cysteine (0.5%; w/v) in DPBS (pH 7.4) was added for 5
minutes at room temperature as a mucolytic agent (Miyamoto and
Bakaletz, Microb. Pathog., 21: 343-356 1996). Fluids were
centrifuged (300.times.g, 5 min) to remove cellular debris, red
blood cells and inflammatory cells, and supernatants containing
bacteria were transferred to a fresh tube. Bacteria were incubated
with chinchilla antiserum (1:50 dilution) directed against a whole
OMP preparation, derived from NTHi strain 86-028NP, for 45 minutes
at 4.degree. C., pelleted by centrifugation (2000.times.g, 5 min)
and washed twice with cold DPBS containing 0.05% bovine serum
albumin. Bacteria were subsequently labeled with cross-reactive
phycoprobe R-PE anti-human IgG (H+L) antibody (10 .mu./ml in 100
.mu.l PBS; Biomeda Corp) for 30 minutes at 4.degree. C. Following
three successive washes to remove unbound antibody, cells were
resuspended in 300 .mu.l DPBS for FACS analysis.
EXAMPLE 6
Identification of Promoters Induced in Vivo in Acute Otitis
Media
[0110] H. influenzae 86-028NP transformed with the promoter trap
library was grown overnight on chocolate agar. To select against
those clones containing promoters that expressed gfp in vitro, the
library was subjected to one round of FACS analysis (as described
in Example 6), collecting only those clones expressing low-level
amounts of GFP. These clones were pooled and used to inoculate the
chinchilla middle ear transbullarly. Following 24 and 48 hours of
infection, bacteria-containing effusions were removed by
epitympanic tap. Bacteria were indirectly labeled with R-PE-labeled
antibody and subjected to FACS analysis by gating on fluorescently
tagged bacteria but sorting for those that were also expressing.
These clones were used to reinfect animals for further enrichment.
Following the final round of sorting, single colony isolates were
screened in vitro for lack of fluorescence.
[0111] Those clones isolated by FACS analysis (positive for GFP
fluorescence in vivo), which did not emit fluorescence in vitro
were prepared for plasmid isolation and identification of insert
DNA sequence. These clones were grown overnight on chocolate agar
plates containing kanamycin and prepared for plasmid isolation
using the Qiaprep Miniprep Kit (Qiagen) according to the
manufacturer's protocol. Plasmid insert DNA was sequenced using the
primer 5'-TGCCCATTAACATCACCATCTA-3' (SEQ ID NO: 588) that is
complementary to the gfpmut.sup.3 gene and downstream of the insert
DNA. Sequencing reactions were performed using the ABI prism
BigDye.RTM. terminator cycle sequencing ready reaction kit (Applied
Biosystems) according to manufacturer's protocol using a GeneAmp
PCR System 9700 (Applied Biosystems). The sequences were then
purified by passage through sephadex G-50 in a 96-well multiscreen
HV plate (Millipore) and subsequently analyzed on an ABI Prism 3100
DNA analyzer (Applied Biosystems).
[0112] Insert sequences were compared to the complete annotated
sequence of H. influenzae strain Rd. Those inserts with no
nucleotide homology to strain Rd were subsequently analyzed using
the BLASTN and BLASTX algorithms. Further sequence analysis was
performed with DNASTAR (Madison, Wis.). Inserts in the correct
orientation and containing sequence 5' to a predicted ORF contained
a putative promoter that was preferentially active when the NTHi
bacteria were in the chinchilla middle ear.
[0113] Fifty-two clones with putative promoters that were regulated
in vivo were isolated. Of the 44 candidate clones containing
sequence similar to that identified in H. influenzae strain Rd,
quantitative comparison of gene expression in vitro and in vivo
confirmed up-regulated gene expression for twenty-six genes (60%)
when NTHi respond to environmental cues present in the chinchilla
middle ear and these genes are summarized in Table 4A below. The in
vivo-regulated promoters driving expression of genes are predicted
to be involved in membrane transport, environmental informational
processing, cellular metabolism, gene regulation, as well as
hypothetical proteins with unknown function.
[0114] In order to confirm the induction of putative promoter
candidates in vivo, the relative amount of messenger RNA expression
was compared when NTHi strain 86-028NP was grown in vitro to
mid-log phase or in vivo for 48 hours. The RNA was isolated using
TRIzol LS reagent (Gibco Life Technologies) according to the
manufacturer's protocol. DNA was removed from the RNA preparation
using DNA-free kit (Ambion) according to the manufacturer's
protocol. DNase I treated RNA samples were purified by passage
through a Qiagen RNeasy column. RNA purity and integrity was
assessed by 260/280 nm spectrophotometer readings and on the
Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.
[0115] In order to independently confirm the FACS data, we
determined the relative expression of candidate genes by
quantitative RT-PCR. The parent strain 86-028NP, was used for these
studies. Real-time quantitative RT-PCR using the one-step
QuantiTect SYBR Green RT-PCR kit (Qiagen) assessed transcription
levels according to the manufacture's instructions. Briefly, using
primers generated to an open reading frame downstream of the
putative in vivo-induced promoters identified by FACS analysis,
gene-specific mRNA was reverse transcribed and amplified by RT-PCR
on the ABI Prism 7700 sequence detection system (Applied
Biosystems). The amount of product was calculated using a standard
curve generated to known amounts of bacterial genomic DNA
(10.sup.2-10.sup.7 genomic copies DNA) by amplifying a fragment of
the gyrase (gyr) gene. Controls were analyzed in parallel to verify
the absence of DNA in the RNA preparation (-RT control) as well as
the absence of primer dimers in control samples lacking template
RNA. In addition, RT-PCR products were analyzed by gel
electrophoresis and, in all cases, a single product was observed at
the appropriate base pair size. Amounts of bacterial RNA between
samples were normalized relative to gyr expression, shown to be
constitutively expressed under various growth conditions that we
tested in vitro. Known amounts of bacterial genomic DNA
(10.sup.2-10.sup.7 genomic copies DNA) were used to generate a
standard curve for RT-PCR quantitation by amplifying a fragment of
the gyrase (gyr) gene. Gyrase is constitutively expressed in vitro
under various growth conditions and was therefore used to normalize
total bacterial RNA levels between samples. Relative gene
expression in vivo was compared to that of gene expression in vitro
and data expressed as fold-increase are summarized in Table 4A.
[0116] The 8-fold sequencing of the NTHi genome identified the full
length open reading frames for the majority of genes listed in
Table 4A. Table 4B provides the full length nucleotide sequence
within the NTHi genome and the corresponding amino acid sequence.
The fold induction of the gene due to environmental cues present in
the chinchilla middle ear and the product or function of the gene
are repeated in Table 4B for convenience. TABLE-US-00006 TABLE 4A
SEQ Gene or ID GenBank Fold Category ORF NO: Protein ID Induction
Product or Function Amino acid hisB 589 NP_438632 2.9 Histidine
biosynthesis metabolism bifunctional protein Lipoprotein lppB 590
NP_438862.1 2.6 Lipoprotein B homologue Membrane transport sapA 591
NP_439780.1 2.8 Peptide ABC transporter; periplasmic SapA precursor
lolA 592 NP_439736.1 2.4 Outer membrane lipoproteins carrier
protein precursor rbsC 593 NP_438661.1 5.1 Ribose transport system
permease protein Purine synthesis purE 594 NP_439757.1 51.7
Phosphoribosylaminoimidazole carboxylase catalytic subunit; PurE
Biosynthetic and ribB 595 NP_438923.1 8.3 3,4-dihydroxy-2-butanone
4- metabolic functions phosphate synthase; riboflavin biosynthesis
arcB 596 NP_438753.1 10 Ornithine carbamolytransferase; arginine
degradation uxuA 597 NP_438228.1 3.1 Mannonate dehydratase;
production of glyceraldehyde 3-phosphate dsbB 598 NP_438589.1 2.6
Disulfide oxidoreductase; disulfide bond formation protein B ureH
599 NP_438693.1 3.9 Urease accessory protein licC 600 NP_439688.1
2.3 Phosphocholine (ChoP) cytidylyltransferase HI1647 601
NP_439789.1 2.0 Putative pyridoxin biosynthesis protein; singlet
oxygen resistance protein DNA replication, ispZ 602 P43810 2.5
Probable intracellular repair septation protein radC 603
NP_439113.1 2.1 DNA repair protein mukF 604 P45185 2.0 MukF protein
homologue; remodeling of nucleiod structure Gene regulation glpR
605 NP_438777.1, 2.8 Glycerol-3-phosphate regulon NP_439170.1
repressor ihfB 606 P43724 2.5 Integration host factor beta subunit
argR 607 NP_439365.1 2.7 Arginine repressor cspD 608 NP_439584.1
2.1 Cold shock like protein; stress response protein Hypothetical
or HI0094 609 NP_438267.1 8.3 Hypothetical protein unknown HI1163
610 NP_439321.1 2.3 Conserved hypothetical proteins protein;
putative oxidase HI1063 611 NP_439221.1 2.7 Hypothetical protein
HI0665 612 NP_438824.1 2.8 Hypothetical protein HI1292 613
NP_439444.1 2.6 Hypothetical protein HI1064 614 NP_439222.1 2.6
Hypothetical protein
[0117] TABLE-US-00007 TABLE 4B Full Gene Length or Nucleotide Amino
Acid Fold Product or Category ORF Sequence Sequence Location in
Contig Induction Function Amino hisB SEQ ID NO: SEQ ID NO: nt.
68378-67290 2.9 Histidine acid 615 616 of SEQ ID NO: biosynthesis
metabolism 680 (contig 13) bifunctional protein Membrane sapA SEQ
ID NO: SEQ ID NO: nt. 200403-198709 2.8 Peptide ABC transport 617
618 of SEQ transporter; ID NO: 685 periplasmic SapA (contig 18)
precursor rbsC SEQ ID NO: SEQ ID NO: nt. 42773-41802 5.1 Ribose
transport 619 620 of SEQ ID NO: system permease 680 (contig 13)
protein Purine purE SEQ ID NO: SEQ ID NO: nt. 219625-219131 51.7
Phosphoribosylaminoimidazole synthesis 621 622 of SEQ carboxylase
catalytic ID NO: 685 subunit; PurE (contig 18) Biosynthetic ribB
SEQ ID NO: SEQ ID NO: nt. 131537-132184 8.3 3,4-dihydroxy-2- and
623 624 of SEQ butanone 4- metabolic ID NO: 682 phosphate synthase;
functions (contig 15) riboflavin biosynthesis arcB SEQ ID NO. SEQ
ID NO: nt. 49710-48706 10 Ornithine 625 626 of SEQ ID NO:
carbamolytransferase; 681 (contig 14) arginine degradation uxuA SEQ
ID NO: SEQ ID NO: nt. 840671-841855 3.1 Mannonate 627 628 of SEQ
dehydratase; ID NO: 685 production of (contig 18) glyceraldehyde 3-
phosphate dsbB SEQ ID NO: SEQ ID NO: nt. 388050-388583 2.6
Disulfide 629 630 of SEQ oxidoreductase; ID NO: 384 disulfide bond
(contig 17) formation protein B ureH SEQ ID NO: SEQ ID NO: nt.
4452-5267 of 3.9 Urease accessory 631 632 SEQ ID NO: 680 protein
(contig 13) licC SEQ ID NO: SEQ ID NO: nt. 355083-354382 2.3
Phosphocholine 633 634 of SEQ (ChoP) ID NO: 385
cytidylyltransferase (contig 18) HI1647 SEQ ID NO: SEQ ID NO: nt.
664017-664892 2.0 Putative pyridoxin 635 636 of SEQ biosynthesis
protein; ID NO: 685 singlet oxygen (contig 18) resistance protein
DNA ispZ SEQ ID NO: SEQ ID NO: nt. 4512-5069 of 2.5 Probable
replication, 637 638 SEQ ID NO: 683 intracellular repair (contig
16) septation protein radC SEQ ID NO: SEQ ID NO: nt. 132695-132030
2.1 DNA repair protein 639 640 of SEQ ID NO: 683 (contig 16) mukF
SEQ ID NO: SEO ID NO: nt. 504549-503215 2.0 MukF protein 641 642 of
SEQ homologue; ID NO: 685 remodeling of (contig 18) nucleiod
structure Gene glpR SEQ ID NO: SEQ ID NO: nt. 72716-73483 2.8
Glycerol-3- regulation 643 644 of SEQ ID NO: phosphate regulon 682
(contig 15) repressor ihfB SEQ ID NO: SEQ ID NO: nt. 661004-660720
2.5 Integration host 645 646 of SEQ factor beta subunit ID NO: 685
(contig 18) argR SEQ ID NO: SEQ ID NO: nt. 178540-178085 2.7
Arginine repressor 647 648 of SEQ ID NO: 685 (contig 18) cspD SEQ
ID NO: SEQ ID NO: nt. 435310-435528 2.1 Cold shock like 649 650 of
SEQ protein; stress ID NO: 685 response protein (contig 18)
Hypothetical HI1163 SEQ ID NO: SEQ ID NO: nt. 137202-134119 2.3
Conserved or 651 652 of SEQ hypothetical protein; unknown ID NO:
685 putative oxidase proteins (contig 18) HI1063 SEQ ID NO: SEQ ID
NO: nt. 35158-34937 2.7 Hypothetical protein 653 654 of SEQ ID NO:
685 (contig 18) HI0665 SEQ ID NO: SEQ ID NO: nt. 17949-18980 2.8
Hypothetical protein 655 656 of SEQ ID NO: 679 (contig 12) HI1292
SEQ ID NO: SEQ ID NO: nt. 555002-555799 2.6 Hypothetical protein
657 658 of SEQ ID NO: 685 (contig 18)
EXAMPLE 7
Identification of Virulence-Associated Genes
[0118] In many bacterial species, a subset of virulence-associated
genes is regulated by errors in replication of short repeats. These
repeats may be 5' to a gene or in the coding sequence, and their
presence is an indication of controlled expression of the gene,
which indicates association with virulence. Addition or deletion of
a repeat results in the expression or of lack of expression of the
particular virulence determinant.
[0119] The NTHi H. influenzae strain 86-028NP contig set was
queried for short oligonucleotide repeats. The region surrounding
the repeats was analyzed to identify the gene(s) associated with
the repeat. Table 5 lists the identified repeats and the ORF
(identified by BLAST) associated with each repeat.
[0120] Further sequence analysis has identified the full length
nucleotide sequence of the virulence-associated genes and the
corresponding amino acid sequences encoded by the ORF. The derived
amino acid sequences are highly homologous to the listed Genbank
sequence. TABLE-US-00008 TABLE 5 Location in Location in Full
Length Amino 3-fold 8-fold Nucleotide Acid Genebank Repeat Contigs
Contigs Sequence Sequence Accession No. SEQ ID 115 nt.
484533-483643 SEQ ID SEQ ID NP_439538.1 NO: 581 nt. 473-540 of NO:
659 NO: 660 of SEQ ID SEQ ID NO: 685 NO: 115 (contig 18) SEQ ID 377
nt. 416274-414910 SEQ ID SEQ ID P45217 NO: 582 nt. 546-597 of NO:
661 NO: 662 of SEQ ID NO: SEQ ID 685 (contig NO: 337 18) SEQ ID 505
nt. 414500-416614 SEQ ID SEQ ID AAK76425 NO: 583 nt. 310-393 of NO:
663 NO: 664 of SEQ ID NO: SEQ ID 684 (contig NO: 505 17) SEQ ID 508
nt. 506516-507913 SEQ ID SEQ ID NP_439520 NO: 584 nt. 2079-2120 of
NO: 665 NO: 666 of SEQ ID NO: SEQ ID 685 (contig NO: 508 18) SEQ ID
518 nt. 354274-352406 SEQ ID SEQ ID NP_284893 NO: 585 nt. 758-789
of NO: 667 NO: 668 of SEQ ID NO: SEQ ID 684 (contig NO: 518 17) SEQ
ID 543 nt. 347864-243236 SEQ ID SEQ ID AAA20524 NO: 586 nt.
1814-196 of NO: 669 NO: 670 of SEQ ID NO: SEQ ID 685 (contig NO:
543 18) SEQ ID 543 nt. 699709-704187 SEQ ID SEQ ID AAD56660 NO: 586
nt. 1814-196 of NO: 671 NO: 672 of SEQ ID NO: SEQ ID 685 (contig
NO: 543 18) SEQ ID 567 nt. 85546-84689 SEQ ID SEQ ID ZP_00053190
NO: 587 nt. 13309-13320 of NO: 673 NO: 674 of SEQ ID NO: SEQ ID 681
(contig NO: 567 14)
EXAMPLE 8
Identification of Unique NTHi Gene Sequences
[0121] Genes associated with NTHi virulence were also identified by
comparing the level of expression of the gene when the NTHi
bacterium was infecting a tissue verses the level of expression of
the same gene when the NTHi was grown on artificial laboratory
media. These novel genes were identified using the promoter trap
techniques described above in Examples 4-6, and subsequently
comparisons with the known Rd genome demonstrated these genes are
unique to NTHi strain 86-028NP.
[0122] The DNA sequence identified using this screening procedure
are set forth as SEQ ID NOS: 577-580. These sequences did not
contain genes or gene fragments that have homologues in the H.
influenzae Rd. genome sequence. Even though these are completely
novel sequences, due to their expression level during NTHi
infection in the chinchilla middle ear, it is likely that
expression of these genes are involved in NTHi virulence.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070264256A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070264256A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References